Integrated Systems and Methods for Diversity Generation and Screening

ABSTRACT

Integrated systems and methods for diversity generation and screening are provided. The systems use common fluid and array handling components to provide nucleic acid diversification, transcription, translation, product screening and subsequent diversification reactions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/154,936, filedMay 23, 2002, which is a continuation of U.S. Ser. No. 09/760,010, filedJan. 10, 2001, which claims the benefit under 35 U.S.C. § 119(e) of USprovisional patent applications INTEGRATED SYSTEMS AND METHODS FORDIVERSITY GENERATION AND SCREENING by Bass et al. U.S. Ser. No.60/175,551 filed Jan. 11, 2000 and INTEGRATED SYSTEMS AND METHODS FORDIVERSITY AND SCREENING by Bass et al. U.S. Ser. No. 60/213,947 filedJun. 23, 2000. Each of the above documents is incorporated herein byreference in their entireties and for all purposes.

FIELD OF THE INVENTION

The present invention relates to automated devices and systems forperforming nucleic acid recombination, mutation, shuffling and otherdiversity generating reactions in vitro, as well as related methods ofperforming automated diversity generation reactions. The devices andsystems can include, e.g., modules for generating diversity in nucleicacids, for recombining these nucleic acids, for arraying the nucleicacids, for making or copying arrays of reaction mixtures comprising thenucleic acids and for performing in vitro translation and/ortranscription of diverse libraries of nucleic acids. Related methods forperforming such shuffling reactions in vitro are also provided.

BACKGROUND OF THE INVENTION

Today's laboratory attempts to meet the dramatically increasing need foranalytical data brought about by the increased pace of new productdevelopment, increased research, demands for stricter quality control,and the like. Labs deliver data in a timely, cost-efficient way whileensuring precise results, clear documentation, and minimal use ofskilled (and, therefore, expensive) personnel. For example, automatedsystems have been proposed to assess a variety of biological phenomena,including, e.g., expression levels of genes in response to selectedstimuli (Service (1998) “Microchips Arrays Put DNA on the Spot” Science282:396-399), high throughput DNA genotyping (Zhang et al. (1999)“Automated and Integrated System for High-Throughput DNA GenotypingDirectly from Blood” Anal. Chem. 71:1138-1145) and many others.Similarly, integrated systems for performing mixing experiments, DNAamplification, DNA sequencing and the like are also available (See,e.g., Service (1998) “Coming Soon: the Pocket DNA Sequencer” Science282: 399-401).

Improvements in laboratory automation continually increase theproductivity of laboratory workers and provide for more precise results,clearer documentation and the like, as compared to the performance ofunautomated tasks. The automation of laboratory procedures using devicesand/or systems dedicated to particular tasks in the laboratorysubstantially enhances the speed and reproducibility of a variety ofexperimental tasks. Product research, regulatory approval and qualitycontrol in industries such as pharmaceuticals, chemicals, andbiotechnology routinely involve the testing of thousands (or evenhundreds of thousands) of samples.

Automated systems typically perform, e.g., repetitive fluid handlingoperations (e.g., pipetting) for transferring material to or fromreagent storage systems such as microtiter trays, which are used asbasic container elements for a variety of automated laboratory methods.Similarly, the systems manipulate, e.g., microtiter trays and control avariety of environmental conditions such as temperature, exposure tolight or air, and the like.

Many such automated systems are commercially available. For example, avariety of automated systems are available from the Zymark Corporation(Zymark Center, Hopkinton, Mass.), which utilize various Zymate systems,which typically include, e.g., robotics and fluid handling modules.Similarly, the common ORCA® robot, which is used in a variety oflaboratory systems, e.g., for microtiter tray manipulation, is alsocommercially available, e.g., from Beckman Coulter, Inc. (Fullerton,Calif.).

More recently, microfluidic systems have established the potential foreven greater automation and laboratory productivity increases. In thesemicrofluidic systems, automated fluid handling and other samplemanipulations are controlled at the microscale level. Such systems arenow commercially available. For example, the Hewlett-Packard (AgilentTechnologies) HP2100 bioanalyzer utilizes LabChip™ technology tomanipulate extremely small sample volumes. In this “lab-on-a-chip,”system, sample preparation, fluid handling and biochemical analysissteps are carried out within the confines of a microchip. The chips havemicrochannels fabricated, e.g., in glass, providing interconnectednetworks of fluid reservoirs and pathways.

While many automated systems are now available, the application ofautomated systems to non-routine sample handling and analysis remainschallenging. In particular, the application of automation to newtechnologies in the field of molecular biology would be desirable. Forexample, some of the most significant new classes of techniques inmolecular biology are found in the field of rapid forced molecularevolution. In rapid evolution processes, diversity is generated innucleic acids of interest via mutation, recombination, or othermechanisms, which are screened for one or more desirable activities, orencoded activities. These processes are repeated until a nucleic acidpossessing or encoding a desired activity level is produced. The presentinvention provides significant new automated systems and methods whichfacilitate nucleic acid shuffling and other diversitygenerating/screening processes of interest.

SUMMARY OF THE INVENTION

The present invention provides automated devices for performing nucleicacid shuffling and other diversity generating reactions in vitro and invivo. The devices can include, e.g., modules for generating diversity innucleic acids, for recombining these nucleic acids, for arraying thenucleic acids, for making or copying arrays of reaction mixturescomprising shuffled mutated or otherwise diversified nucleic acids andfor performing in vitro translation and/or transcription of diverselibraries of nucleic acids (including in an array-based format). Relatedmethods for performing automated mutation, recombination and/orshuffling reactions in vitro and in vivo are also provided.

For example, the present invention comprises, e.g., devices and/orintegrated systems which include a physical or logical array of reactionmixtures. The reaction mixtures include one or more diversified (e.g.,shuffled or mutagenized) nucleic acids and/or one or more transcribedshuffled or transcribed mutagenized nucleic acids and one or more invitro transcription and/or translation reagents. A variety of variantforms and implementations of these devices/integrated systems, as wellas related methods are described herein.

The devices and integrated systems optionally include any of a varietyof component or module elements. These can include, e.g., one or moreduplicates of the physical or logical array. A bar-code based sampletracking module, which includes a bar code reader and a computerreadable database comprising at least one entry for at least one arrayor at least one array member can also be included, in which the entry iscorresponded to at least one bar code. The device or integrated systemcan include a long term storage device such as a refrigerator; anelectrically powered cooling device, a device capable of maintaining atemperature of <0 C, a freezer, a device which uses liquid nitrogen orliquid helium for cooling storing or freezing samples, a containercomprising wet or dry ice, a constant temperature and/or constanthumidity chamber or incubator; or an automated sample storage orretrieval unit. The device or integrated can also include one or moremodules for moving arrays or array members into the long term storagedevice.

The device or integrated system can, and often do, include a copy arraycomprising a copy of each of a plurality of members of the one or moreshuffled or mutagenized nucleic acids in a physically or logicallyaccessible arrangement of the members. A plurality of the reactionmixtures can include one or more translation products or one or moretranscription products, or both one or more translation products and oneor more transcription products. The array of reaction mixtures can be ina solid phase, liquid phase or mixed phase array which includes one ormore of: the one or more shuffled or mutated nucleic acids, the one ormore transcribed shuffled nucleic acids, and the one or more in vitrotranslation reagents. The one or more shuffled or mutated nucleic acidsare optionally homologous or heterologous. The one or more transcribedshuffled or mutated nucleic acid(s) typically, though not necessarily,includes an mRNA.

The one or more in vitro translation reagents which are optionallypresent in the array typically include transcription reagents, e.g.,reticulocyte lysates, rabbit reticulocyte lysates, canine microsometranslation mixtures, wheat germ in vitro translation (IVT) mixtures, E.coli lysates, or the like. As already noted, the arrays optionallyfurther include one or more in vitro transcription reagents, such as anE. coli lysate, an E. coli extract, an E. coli s20 extract, a caninemicrosome system, a HeLa nuclear extract in vitro transcriptioncomponent, an SP6 polymerase, a T3 polymerase a T7 RNA polymerase, orthe like.

The device or integrated system can include a nucleic acid shuffling ormutagenesis module, which accepts input nucleic acids or characterstrings corresponding to input nucleic acids and manipulates the inputnucleic acids or the character strings corresponding to input nucleicacids to produce output nucleic acids, which include the one or moreshuffled or mutagenized nucleic acids in the reaction mixture array. Theoutput nucleic acids optionally comprise one or more sequence whichcontrols transcription or translation. Such modules include a DNAshuffling module, which accepts input DNAs or character stringscorresponding to input DNAs and manipulates the input DNAs or thecharacter strings corresponding to input DNAs to produce output DNAs,which output DNAs include the one or more shuffled DNAs in the reactionmixture array. The nucleic acid shuffling or mutagenesis module isoptionally preceded in the system or device by a module which allowsoverlapping synthetic oligonucleotides to be first assembled intooligonucleotide multimers or functional open reading frames prior toentering the mutagenesis or shuffling module. The module(s) can beoperatively linked to or include a thermocycling device, or amutagenesis module. In one aspect, the nucleic acid shuffling ormutagenesis module fragments the input nucleic acids to produce nucleicacid fragments. Alternately, the input nucleic acids optionally includecleaved or synthetic nucleic acid fragments. Optionally, the shufflingor mutagenesis module is mechanically, electronically, robotically orfluidically coupled to at least one other array operation module. Thenucleic acid shuffling or mutagenesis module can perform any of avariety of operations, including PCR, StEP PCR, uracil incorporation,chain termination, or the like. Optionally, the nucleic acid shufflingmodule separates, identifies, purifies or immobilizes any productelongated nucleic acid.

The nucleic acid shuffling module optionally includes an identificationportion which identifies one or more nucleic acid portion or subportion(e.g., by sequencing or any other product deconvolution method).Similarly, the nucleic acid shuffling module optionally includes afragment length purification portion which purifies selected lengthfragments of the nucleic acid fragments. In one embodiment, the nucleicacid shuffling module permits hybridization of the nucleic acidfragments. The module can also include a polymerase which elongates thehybridized nucleic acid.

The module can control incorporation of features into product nucleicacids. For example, the nucleic acid shuffling module can combine one ormore translation or transcription control sequence into elongatedproduct nucleic acids. The translation or transcription controlsequence(s) can be combined into the elongated nucleic acid using thepolymerase, or a ligase, or both. The nucleic acid shuffling moduleoptionally determines a recombination frequency or a length, or both arecombination frequency and a length, for any product nucleic acid(s).Similarly, the nucleic acid shuffling module can determine nucleic acidlength by detecting incorporation of one or more labeled nucleic acid ornucleotide into the resulting elongated nucleic acid. For example, thenucleic acid shuffling module optionally determines nucleic acid lengthby detecting one or more label (e.g., dye, radioactive label, biotin,digoxin, or a fluorophore) associated with any product nucleic acid. Forexample, the nucleic acid shuffling module can determine nucleic acidlength with a fluorogenic 5′ nuclease assay.

The devices and integrated systems can utilize conventional ormicroscale construction. Thus, in one aspect, the physical or logicalarray of reaction mixtures is optionally incorporated into a microscaledevice, or at least one of the reaction mixtures is incorporated into amicroscale device, or the one or more shuffled or mutagenized nucleicacids or the one or more transcribed shuffled or mutagenized nucleicacids is found within a microscale device, or the one or more in vitrotranslation reagents is optionally found within a microscale device. Thenucleic acid shuffling module optionally comprises one or moremicroscale channel (e.g., a microcapillary or chip) through which ashuffling reagent or product is flowed. Liquid flow through the deviceis mediated, e.g., by capillary flow, differential pressure between oneor more inlets and outlets, electroosmosis, hydraulic or mechanicalpressure, or peristalsis.

Nucleic acid fragments for use in the systems and devices of theinvention are optionally contacted in a single pool, or in multiplepools. For example, the nucleic acid shuffling module optionallydispenses the resulting elongated nucleic acids into one or moremultiwell plates, or onto one or more solid substrates, or into one ormore microscale systems, or into one or more containers. The nucleicacid shuffling module optionally pre-dilutes any product nucleic acidsand dispenses them into one or more multiwell plates, e.g., at aselected density per well of the product nucleic acid(s).

For example, in one embodiment, the nucleic acid shuffling moduledispenses elongated nucleic acids into one or more master multiwellplates and/or PCR amplifies the resulting master array of elongatednucleic acids to produce an amplified array of elongated nucleic acids.Optionally, the module includes a array copy system which transfersaliquots from the wells of the one or more master multiwell plates toone or more copy multiwell plates. The array of reaction mixtures isoptionally formed by separate or simultaneous addition of an in vitrotranscription reagent and an in vitro translation reagent to the one ormore copy multiwell plates, or to a duplicate set thereof.

In one embodiment, the device or integrated system, further includes oneor more sources of one or more nucleic acids. The one or more sourcescollectively or individually can include a first population of nucleicacids, wherein shuffled or mutant nucleic acids are produced byrecombining the one or more members of the first population of nucleicacids. The one or more sources of nucleic acids include, e.g., at leastone nucleic acid selected from: a synthetic nucleic acid, a DNA, an RNA,a DNA analogue, an RNA analogue, a genomic DNA, a cDNA, an mRNA, a DNAgenerated by reverse transcription, an nRNA, an aptamer, a polysomeassociated nucleic acid, a cloned nucleic acid, a cloned DNA, a clonedRNA, a plasmid DNA, a phagemid DNA, a viral DNA, a viral RNA, a YAC DNA,a cosmid DNA, a fosmid DNA, a BAC DNA, a P1-mid, a phage DNA, asingle-stranded DNA, a double-stranded DNA, a branched DNA, a catalyticnucleic acid, an antisense nucleic acid, an in vitro amplified nucleicacid, a PCR amplified nucleic acid, an LCR amplified nucleic acid, aQ∃-replicase amplified nucleic acid, an oligonucleotide, a nucleic acidfragment, a restriction fragment and a combination thereof.

The device or integrated system optionally includes a populationdestination region, wherein, during operation of the device, one or moremembers of the first population are moved from the one or more sourcesof the one or more nucleic acids to the one or more destination regions(e.g., in the form of a solid phase array, a liquid phase array, acontainer, a microtiter tray, a microtiter tray well, a microfluidiccomponent, a microfluidic chip, a test tube, a centrifugal rotor, amicroscope slide, an organism, a cell, a tissue, a liposome, a detergentparticle, or any combination thereof). Thus, the device or integratedsystem can include nucleic acid movement means (e.g., a fluid pressuremodulator, an electrokinetic fluid force modulator, a thermokineticmodulator, a capillary flow mechanism, a centrifugal force modulator, arobotic armature, a pipettor, a conveyor mechanism, a peristaltic pumpor mechanism, a magnetic field generator, an electric field generator,one or more fluid flow path, etc.) for moving the one or more membersfrom the one or more sources of the one or more nucleic acids to the oneor more destination regions (for example, nucleic acids to be recombinedcan be moved into contact with one another). During operation of thedevice, the in vitro transcription reagent or an in vitro translationreagent is typically flowed into contact with the members of the firstpopulation. Optionally, members of the first population are fixed(immobilized) at the one or more sources of one or more nucleic acids orat the one or more destination regions. During operation of the device,the first population of nucleic acids is optionally arranged into one ormore physical or logical recombinant nucleic acid arrays, which areoptionally duplicated.

The device or integrated system can include one or more reaction mixturearraying modules which move one or more of the one or more shuffled (ormutated) nucleic acids or the one or more transcribed shuffled ormutated nucleic acids or the in vitro translation reactant componentsinto one or more selected spatial positions. This places the one or moreshuffled mutated or otherwise diversified nucleic acids or the one ormore transcribed shuffled or otherwise diversified nucleic acids or thein vitro translation reactant component into one or more locations inthe array of reaction mixtures. Thus, this module can be used togenerate a recombined/mutated/shuffled nucleic acid master or duplicatearray which physically or logically corresponds to positions of mutated,shuffled or other product nucleic acids in a reaction mixture array. Thedevice or integrated system can include a nucleic acid amplificationmodule, which module amplifies members of the mutated or shufflednucleic acid master array, or a duplicate thereof. The arraying andamplification modules can be integrated in one module or device.

The amplification module can include a heating or cooling element (e.g.,to perform PCR, LCR or the like). For example, in one embodiment, theamplification module includes a DNA micro-amplifier. For example, themicro-amplifier can include a programmable resistor, a micromachinedzone heating chemical amplifier, a Peltier solid state heat pump, a heatpump, a heat exchanger, a hot air blower, a resistive heater, arefrigeration unit, a heat sink, a Joule Thompson cooling device, or anycombination thereof. The arraying/amplification module can produce aduplicate amplified array which produces amplicons of the nucleic acidmaster array, or duplicates thereof.

During operation of the overall device or system, the array of reactionmixtures produces an array of reaction mixture products. The device orintegrated system can include one or more product identification orpurification modules, which product identification modules identify oneor more members of the array of reaction products. For example, productidentification or purification modules can include one or more of: agel, a polymeric solution, a liposome, a microemulsion, a microdroplet,an affinity matrix, a plasmon resonance detector, a BIACORE, a GCdetector, an ultraviolet or visible light sensor, an epifluorescencedetector, a fluorescence detector, a fluorescent array, a CCD, a digitalimager, a scanner, a confocal imaging device, an optical sensor, a FACSdetector, a micro-FACS unit, a temperature sensor, a mass spectrometer,a stereo-specific product detector, an Elisa reagent, an enzyme, anenzyme substrate an antibody, an antigen, a refractive index detector, apolarimeter, a pH detector, a pH-stat device, an ion selective sensor, acalorimeter, a film, a radiation sensor, a Geiger counter, ascintillation counter, a particle counter, an H2O2 detection system, anelectrochemical sensor, ion/gas selective electrodes, or a capillaryelectrophoresis element. For ease of detection, the one or more reactionproduct array members are optionally moved into proximity to the productidentification module, or the product identification module can performan xyz translation, thereby moving the product identification moduleproximal to the array of reaction products. Similarly, the one or morereaction product array members are optionally flowed into proximity tothe product identification module, where an in-line purification systempurifies the one or more reaction product array members from associatedmaterials.

Typical reaction products include, e.g., one or more polypeptide, one ormore nucleic acid, one or more catalytic RNA (e.g., a ribozyme), or oneor more biologically active RNA (e.g., an anti-sense RNA). In one classof embodiments, the device or integrated system can include a source ofone or more lipid which is flowed into contact with the one or morepolypeptide, or into contact with the physical or logical array ofreaction mixtures, or into contact with the one or more transcribedshuffled or mutagenized nucleic acids, thereby producing one or moreliposomes or micelles comprising the polypeptide, reaction mixturecomponents, or one or more transcribed shuffled or mutagenized nucleicacids. The reaction products can include one or more polypeptide whichcan be further modified by the system, e.g., by incubation with one ormore protein refolding reagent. For example, refolding agents such asguanidine, guanidinium, urea, detergents, chelating agents, DTT, DTE,chaperonins and the like can be flowed into contact with the protein ofinterest.

Product identification or purification modules in the device orintegrated system can include a protein detector, a protein purificationmeans, or the like. The product identification or purification modulescan also include an instruction set for discriminating between membersof the array of reaction products based upon, e.g., a physicalcharacteristic of the members, an activity of the members,concentrations of the members, or combinations thereof.

The device or integrated system can include a secondary product arrayproduced by re-arraying members of the reaction product array such thatthe secondary product array has a selected concentration of productmembers in the secondary product array. The selected concentration isoptionally approximately the same for a plurality of product members inthe secondary product array. This facilitates comparison of activity ordetectable feature levels across or among members of the secondaryproduct array. In an alternate or complementary aspect, the device orintegrated system can include an instruction set or physical or logicalfilter for determining a correction factor which accounts for variationin polypeptide concentration at different positions in the amplifiedphysical or logical array of polypeptides.

The device or integrated system of can include a substrate additionmodule which adds one or more substrate to a plurality of members of theproduct array or the secondary product array. In this embodiment, asubstrate conversion detector is provided to monitor formation of aproduct produced by contact between the one or more substrate and one ormore of the plurality of members of the product array or the secondaryproduct array. Formation of product or disappearance of substrate ismonitored directly or indirectly, for example, by monitoring loss of thesubstrate or formation of product over time. Formation of the product ordisappearance of substrate is optionally monitored enantioselectively,regioselectively or stereo selectively. For example, formation of theproduct or disappearance of substrate is optionally monitored by addingat least one isomer, enantiomer or stereoismer in substantially pureform (e.g., independent of other potential isomers). Formation of theproduct is optionally monitored by detecting any detectable product,e.g., by monitoring formation of peroxide, protons, or halides, orreduced or oxidized cofactors, changes in heat or entropy which resultfrom contact between the substrate and the product, changes in mass,charge, fluorescence, epifluorescence, by chromatography, luminescenceor absorbance, of the substrate or the product, which result fromcontact between the substrate and the product.

The device or integrated system optionally includes an arraycorrespondence module, which identifies, determines or records thelocation of an identified product in the array of reaction mixtureproducts which is identified by the one or more product identificationmodules, or which array correspondence module determines or records thelocation of at least a first nucleic acid member of the shuffled ormutant nucleic acid master array, or a duplicate thereof, or of anamplified duplicate array, where the member corresponds to the locationof one or more member of the array of reaction products.

The device or integrated system optionally includes one or moresecondary selection module which selects at least the first member forfurther recombination, which selection is based upon the location of aproduct identified by the product identification module(s).

The device or integrated system optionally includes a screening orselection module. For example, the module can include one or more of: anarray reader, which detects one or more member of the array of reactionproducts; an enzyme which converts one or more member of the array ofreaction products into one or more detectable products; a substratewhich is converted by the one or more member of the array of reactionproducts into one or more detectable products; a cell which produces adetectable signal upon incubation with the one or more member of thearray of reaction products; a reporter gene which is induced by one ormore member of the array of reaction products; a promoter which isinduced by one or more member of the array of reaction products, whichpromoter directs expression of one or more detectable products; and anenzyme or receptor cascade which is induced by the one or more member ofthe array of reaction products.

The device or integrated system can include a secondary recombinationmodule, which physically contacts the first member, or an ampliconthereof, to an additional member of the shuffled or mutant nucleic acidmaster array, or the duplicate thereof, or the amplified duplicatearray, thereby permitting physical recombination between the first andadditional members.

The device or integrated system optionally includes a DNA fragmentationmodule which can include a recombination region. The DNA fragmentationmodule can include, e.g., one or more of: a nuclease, a mechanicalshearing device, a polymerase, a random primer, a directed primer, anucleic acid cleavage reagent, a chemical nucleic acid chain terminator,and an oligonucleotide synthesizer. During operation of the device,fragmented DNAs produced in the DNA fragmentation module are optionallyrecombined in the recombination region to produce one or more mutated,shuffled or otherwise altered nucleic acids.

Common operations for the device or system include modules which performone or more of: error prone PCR, site saturation mutagenesis, orsite-directed mutagenesis. Many other diversity generating reactionswhich can be practiced in modules of the devices or systems are setforth herein.

The device or integrated system optionally includes a data structureembodied in a computer, such as an analog computer or a digitalcomputer, or in a computer readable medium. The data structurecorresponds to the one or more shuffled or otherwise modified nucleicacid(s).

The device or integrated system optionally includes one or more reactionmixtures which include one or more mutated or shuffled nucleic acidsarranged in a microtiter tray at an average of approximately 0.1-100shuffled or otherwise modified nucleic acids per well, e.g., an averageof approximately 1-5 such nucleic acids per well.

The device or integrated system optionally includes a diluter whichpre-dilutes the concentration of the one or more shuffled, modified ormutated nucleic acids prior to addition of the shuffled or mutantnucleic acids to the reaction mixtures. The concentration of the one ormore modified, mutated or shuffled nucleic acids after pre-dilution isabout 0.01 to 100 molecules per microliter.

In one class of embodiments, the reaction mixtures are produced in thedevice or system by adding the in vitro translation reactant and,optionally, an in vitro transcription reagent, to a duplicate shuffledor mutated nucleic acid array. The duplicate shuffled or mutated nucleicacid array is duplicated from a master array of the shuffled or mutatednucleic acids produced by spatially or logically separating members of apopulation of the shuffled or mutated nucleic acids to produce aphysical or logical array of the shuffled or mutated nucleic acids. Forexample, the array can be produced by one or more arraying technique,including (1) lyophilizing members of the population of mutated,shuffled or otherwise altered nucleic acids on a solid surface, therebyforming a solid phase array, (2) chemically coupling members of thepopulation of mutated, shuffled or otherwise altered nucleic acids to asolid surface, thereby forming a solid phase array, (3) rehydratingmembers of the population of mutated, shuffled or otherwise alterednucleic acids on a solid surface, thereby forming a liquid phase array,(4) cleaving chemically coupled members of the population of mutated,shuffled or otherwise altered nucleic acids from a solid surface,thereby forming a liquid phase array, (5) accessing one or morephysically separated logical array members from one or more sources ofmutated, shuffled or otherwise altered nucleic acids and flowing thephysically separated logical array members to one or more destination,the one or more destinations constituting a logical array of themutated, shuffled or otherwise altered nucleic acids, and (6) printingmembers of a population of mutated, shuffled or otherwise alterednucleic acids onto a solid material to form a solid phase array.Optionally, greater than about 1% of the physical or logical array ofreaction mixtures comprise shuffled or mutant nucleic acids having oneor more base changes relative to a parental nucleic acid.

In one aspect, one or more mutated, recombined (e.g., shuffled) orotherwise modified nucleic acids are produced by synthesizing a set ofoverlapping oligonucleotides, or by cleaving a plurality of homologousnucleic acids to produce a set of cleaved homologous nucleic acids, orboth, and permitting recombination to occur between the set ofoverlapping oligonucleotides, the set of cleaved homologous nucleicacids, or both the set of overlapping oligonucleotides and the set ofcleaved homologous nucleic acids.

In one aspect, the invention provides a diversity generation device. Thedevice includes a programmed thermocycler and a fragmentation moduleoperably coupled to the programmed thermocycler. The programmedthermocycler typically includes a thermocycler operably coupled to acomputer which includes one or more instruction set, e.g., forcalculating an amount of uracil and an amount of thymidine for use inthe programmed thermocycler, calculating one or more crossover regionbetween two or more parental nucleotides calculating an annealingtemperature, calculating an extension temperature, selecting one or moreparental nucleic acid sequence, or the like.

The one or more instruction set receives user input data and sets up oneor more cycle to be performed by the programmed thermocycler. The inputdata typically includes one or more parental nucleic acid sequence, adesired crossover frequency, an extension temperature, and/or anannealing temperature, or other features which control the reaction ofinterest.

In one aspect, the one or more instruction set calculates an amount ofuracil and an amount of thymidine based on a desired fragment size. Inother aspects, the one or more instruction set directs the one or morecycle on the diversity generation device, e.g., amplifies the one ormore parental nucleic acid sequence, fragments the one or more parentalnucleic acid sequence to produce one or more nucleic acid fragment,reassembles the one or more nucleic acid fragment to produce one or moremutated, shuffled or otherwise altered nucleic acid, and/or amplifiesthe one or more mutated, shuffled or otherwise altered nucleic acid. Forexample, the set can direct amplifying the one or more parental nucleicacid sequence in the presence of uracil. Optionally, the one or morecycle pauses between steps to allow addition of one or morefragmentation reagent.

The one or more instruction set optionally performs one or morecalculation based on one or more theoretical prediction of a nucleicacid melting temperature or on one or more set of empirical data, whichempirical data comprises a comparison of one or more nucleic acidmelting temperature. The one or more instruction set optionallyinstructs the fragmentation module to fragment the parental nucleicacids to produce one or more nucleic acid fragments having a desiredmean fragment size.

The programmed thermocycler comprises a thermocycler and, optionally,software for performing one or more shuffling calculations, whichsoftware is embodied on a web page, an attached computer, an intranetserver, or, e.g., installed directly in the thermocycler.

In one aspect, a similar diversity generation device is provided. Thedevice includes a computer, which includes at least a first instructionset for creating one or more nucleic acid fragment sequence from one ormore parental nucleic acid sequence and a synthesizer module, whichsynthesizes the one or more nucleic acid fragment sequence. The devicealso includes a thermocycler which generates one or more diversesequence from the one or more nucleic acid fragment sequence. The firstinstruction set optionally limits or expands diversity of the one ormore nucleic acid fragment sequence by adding or removing one or moreamino acid having similar diversity; selecting a frequently used aminoacid at one or more specific position; using one or more sequenceactivity calculation; using a calculated overlap with one or moreadditional oligonucleotide; based on an amount of degeneracy, or basedon a melting temperature. In one aspect, the thermocycler performs anassembly/rescue PCR reaction.

The diversity generation device can include a synthesizer module havinga microarray oligonucleotide synthesizer. For example, the synthesizermodule optionally includes an ink-jet printer head based oligonucleotidesynthesizer. The synthesizer module optionally synthesizes the one ormore nucleic acid fragment sequences on a solid support. The synthesizermodule optionally uses one or more mononucleotide coupling reactions orone or more trinucleotide coupling reactions to synthesize the one ormore nucleic acid fragment sequence.

The computer optionally comprises at least a second instruction set,which second instruction set determines at least a first set ofconditions for the assembly/rescue PCR reaction.

The device optionally further includes a screening module for screeningthe one or more diverse sequence for a desired characteristic. Forexample, the screening module optionally comprises a high-throughputscreening module.

In a related aspect, a diversity generation kit is provided. Forexample, the kit can include the diversity generation devices above andone or more reagent for diversity generation. Example reagents includeE. coli, a PCR reaction mixture comprising a mixture of uracil andthymidine, one or more uracil cleaving enzyme, and a PCR reactionmixture comprising standard dNTPs. The one or more uracil cleavingenzyme optionally includes a uracil glycosidase and an endonuclease. Themixture of uracil and thymidine comprises a desired ratio of uracil tothymidine, which desired ratio is calculated by the diversity generationdevice, based upon user selected inputs.

Optionally, the diversity generation kit can include one or moreartificially evolved enzyme such as an artificially evolved polymerase.The kit can also include, e.g., packaging materials, a container adaptedto receive the device or reagents, and instructional materials for useof the device.

The devices and integrated systems herein can include data trackingmodules such as a bar-code based sample tracking module, which includes,e.g., a bar code reader and a computer readable database comprising atleast one entry for at least one array or at least one array member,which entry is corresponded to at least one bar code. Long term storagedevices can also be incorporated into the devices and integrated systemsherein (and the methods herein can include storage in such long termstorage modules). For example, as noted, the storage module can include,e.g., a refrigerator, an electrically powered cooling device, a devicecapable of maintaining a temperature of <0 C; a freezer, a device whichuses liquid nitrogen or liquid helium for cooling storing or freezingsamples, a container comprising wet or dry ice, a constant temperatureand/or constant humidity chamber or incubator, an automated samplestorage or retrieval unit, a dessicator or moisture minimizing orreducing device, one or more modules for moving arrays or array membersinto the long term storage device etc.

As noted in more detail herein, the invention provides devices andintegrated systems, e.g., which include a physical or logical array ofreaction mixtures, each reaction mixture comprising one or more shuffledor mutagenized nucleic acids and one or more transcribed shuffled ortranscribed mutagenized nucleic acids or one or more in vitrotranslation reagents. Also provided are libraries of shuffled or mutatedor mutagenized nucleic acids formatted in a logical and physical arraybased on at least one physical and one activity parameter. Devices orintegrated systems which use a fluorescent or visible signal to sort ashuffled or mutagenized nucleic acid library into a spatial array ofcells, particles or molecules are also provided. These include, e.g., aphysical or logical array of comprising one or more shuffled ormutagenized nucleic acids or one or more transcribed shuffled ortranscribed mutagenized nucleic acids or one or more in vitrotranslation reagents.

The present invention also provides a number of related methods, bothfor use with the integrated systems and devices of the invention and foruse separate from the devices and systems.

For example, in one class of methods of the invention, methods ofprocessing shuffled or mutagenized nucleic acids are provided. In themethods, a physical (e.g., solid or liquid phase) or logical array ofreaction mixtures is provided. A plurality of the reaction mixturesinclude one or more member of a first population of nucleic acids. Thefirst population of nucleic acids include one or more shuffled ormutagenized nucleic acids, or one or more transcribed shuffled ormutagenized nucleic acids. A plurality of the plurality of reactionmixtures typically further include an in vitro translation reactant. Oneor more in vitro translation products produced by a plurality of membersof the physical or logical array of reaction mixtures is then detected.Any of the various array configurations noted above or herein for thedevices and integrated systems of the invention are can be used in thesemethods.

For example, in one embodiment, a population of nucleic acids (which canbe homologous or heterologous) is physically arrayed on a solidsubstrate, such as a chip, slide, membrane, or well of a microtiter trayor plate. The arrayed nucleic acids are recombined with one or moreadditional nucleic acids, thereby providing an arrayed library ofrecombinant nucleic acids. These recombinant nucleic acids are thenamplified and screened to identify members of the array that possess adesired property. In some embodiments, an oligonucleotide primer istethered to the solid substrate and an additional single-strandednucleic acid is annealed to the oligonucleotide which is then extendedwith a nucleic acid polymerase. In alternative embodiments, asingle-stranded template polynucleotide is hybridized with a set ofpartially overlapping complementary nucleic acid fragments which areextended to produce an arrayed library of recombinant nucleic acids. Forexample, one or more template nucleic acids are immobilized on a solidsupport. Partially overlapping complementary nucleic acid fragments areannealed to the template polynucleotide, and extended or ligated toproduce a heteroduplex comprising the template nucleic acid and asubstantially full-length heterolog complementary to the templatenucleic acid. The heterolog is recovered and, optionally, furtherdiversified.

A number of variants of this basic methodology are set forth herein, asare a variety of products produced by the methods and their variants andapparatus and kits for performing the methods.

For example, the one or more mutated, shuffled or otherwise alterednucleic acids are optionally produced in an automatic DNA shuffling,recombination, or mutation module. Optionally, the method includesinputting DNAs or character strings corresponding to input DNAs into theDNA shuffling module and accepting output DNAs from the DNA shufflingmodule, where the output DNAs include the one or more mutated, shuffledor otherwise altered nucleic acids in the reaction mixture array. Theinput DNA in the DNA shuffling module can be cleaved to produce DNAfragments, or provide the input DNAs can include cleaved or syntheticDNA fragments. DNA fragments, e.g., of a selected length can be purifiedin the DNA shuffling module. Purified DNA fragments can be hybridizedand elongated with a polymerase. The resulting elongated nucleic acidscan be separated, identified, cloned, purified, or the like. Arecombination frequency or a length, or both a recombination frequencyand a length for the resulting elongated DNAs can be determined, e.g.,by detecting incorporation of one or more labeled nucleic acid ornucleotide into the elongated DNAs.

The invention provides for a variety of physical manipulations of thevarious reagents and products of the invention, including, flowing,e.g., a shuffling reagent or product through a microscale channel in theDNA shuffling module, contacting the components in single or multiplepools, dispensing materials into one or more multiwell plates,dispensing materials into one or more multiwell plates at a selecteddensity per well of the elongated DNAs, dispensing the product elongatedDNAs into one or more master multiwell plates and PCR amplifying theresulting master array of elongated nucleic acids to produce anamplified array of elongated nucleic acids, etc. Optionally, theshuffling module includes an array copy system which transfers aliquotsfrom the wells of the one or more master multiwell plates to one or morecopy multiwell plates.

The methods optionally include determining an extent of PCRamplification by any available technique, including, e.g., incorporationof a label into one or more amplified elongated nucleic acid, applying afluorogenic 5′ nuclease assay or the like.

In one aspect, the array of reaction mixtures is formed by separate orsimultaneous addition of in vitro transcription reagents and an in vitrotranslation reactant to the one or more copy multiwell plates, or to aduplicate set thereof, wherein the elongated DNAs comprise the one ormore mutated, shuffled or otherwise altered nucleic acids. Typically,the array of reaction mixtures produces an array of reaction mixtureproducts, e.g., comprising one or more polypeptide. The methodsoptionally include re-folding the one or more polypeptide by contactingthe one or more polypeptide with a refolding reagent such as guanidine,urea, DTT, DTE, and/or a chaperonin. The one or more polypeptide withone or more lipid to produce one or more liposome or micelle, whichliposome or micelle comprises the one or more polypeptide.

The methods optionally include moving the one or more reaction productarray members into proximity to a product identification module, ormoving a product identification module into proximity to the reactionproduct array members. The one or more reaction product array membersare optionally flowed into proximity to a product identification module.In-line purification of the one or more reaction product array memberscan be performed.

In one aspect, the method further includes reading the array of reactionmixture products with an array reader which detects one or more memberof the array of reaction products. In another aspect, one or more memberof the array of reaction products is converted with an enzyme into oneor more detectable products. Similarly, one or more substrates can beconverted by the one or more member of the array of reaction productsinto one or more detectable products. These detectable products areoptionally detected in he array reader.

A cell can be contacted to one or more member of the array of reactionproducts, which cell or reaction product, or both, produce a detectablesignal upon contacting the one or more member of the array of reactionproducts.

A variety of detectable events can be induced, including inducing areporter gene with one or more member of the array of reaction products,inducing a promoter with one or more member of the array of reactionproducts which directs expression of one or more detectable products,including inducing an enzyme or receptor cascade with one or more memberof the array of reaction products which is induced by the one or moremember of the array of reaction products.

Methods of recombining members of a physical or logical array of nucleicacids are also provided. In the methods, a first population of nucleicacids is provided, or a data structure (e.g., embodied in a computer, ananalog computer, a digital computer, or a computer readable medium)comprising character strings corresponding to the first population ofnucleic acids (e.g., embodied in a computer, an analog computer, adigital computer, or a computer readable medium) is provided. One ormore members of the first population of nucleic acids are recombined,thereby providing a first population of recombinant nucleic acids.Alternatively, one or more character strings corresponding to one ormore members of the first population of nucleic acids are recombined,thereby providing a population of character strings corresponding to thefirst population of recombinant nucleic acids. In this embodiment, thepopulation of character strings corresponding to the first population ofrecombinant nucleic acids is converted into the first population ofrecombinant nucleic acids, thereby providing the first population ofrecombinant nucleic acids. In either case, members of the population ofrecombinant nucleic acids are spatially or logically separated toproduce a physical or logical array of recombinant nucleic acids. Therecombinant nucleic acids in the physical or logical array ofrecombinant nucleic acids are amplified in vitro (e.g., by enzymatic orsynthetic means) to provide an amplified physical or logical array ofrecombinant nucleic acids. Alternately, members of the population ofrecombinant nucleic acids are amplified (or synthesized) and physicallyor logically separated to produce an amplified physical or logical arrayof recombinant nucleic acids. Typically, the amplified physical orlogical array of recombinant nucleic acids, or a duplicate thereof, isscreened for one or more desired property. Optionally, the amplifiedphysical or logical array of recombinant nucleic acids, or a duplicatethereof, is screened for a desired property. A variety of variants ofthis basic class of methods are set forth herein, as are a variety ofproducts produced by the methods and their variants and kits andapparatus for practicing the methods.

Spatially or logically separating members of the population ofrecombinant nucleic acids to produce a physical or logical array ofrecombinant nucleic acids or amplified recombinant nucleic acidsoptionally includes plating the nucleic acids in a microtiter tray at anaverage of approximately 0.1-10 (e.g., 1-5) array members per well.Optionally, spatially or logically separating the members of thepopulation of recombinant nucleic acids includes diluting the members ofthe population with a buffer. The concentration of the population ofrecombinant nucleic acids after dilution is typically about 0.01 to 100molecules per microliter.

Spatially or logically separating members of the population ofrecombinant nucleic acids to produce a physical or logical array ofrecombinant nucleic acids can also include one or more of: (i)lyophilizing members of the population of recombinant nucleic acids on asolid surface, thereby forming a solid phase array; (ii) chemicallycoupling members of the population of recombinant nucleic acids to asolid surface, thereby forming a solid phase array; (iii) rehydratingmembers of the population of recombinant nucleic acids on a solidsurface, thereby forming a liquid phase array; (iv) cleaving chemicallycoupled members of the population of recombinant nucleic acids from asolid surface, thereby forming a liquid phase array; and, (v) accessingone or more physically separated logical array members from one or moresources of recombinant nucleic acids and flowing the physicallyseparated logical array members to one or more destination.

Methods of recombining members of a physical or logical array of nucleicacid are provided. In the methods, at least a first population ofnucleic acids is arranged in a physical or logical array. One or moremembers of the first population of nucleic acids is recombined with oneor more additional nucleic acid, thereby providing a first physical orlogical array comprising a population of recombined nucleic acids. Therecombined nucleic acids in the physical or logical array of recombinednucleic acids are amplified, usually in vitro, to provide an amplifiedphysical or logical array of recombined nucleic acids. The first oramplified physical or logical array of recombined nucleic acids, or oneor more duplicate thereof, is then screened for one or more desiredproperties. As above, a number of variants of this basic class ofmethods are set forth herein. In some embodiments, the recombination ofnucleic acids is performed on a solid substrate such as a slide,membrane or “chip.” For example, a population of nucleic acids isphysically arrayed on a solid substrate, such as a chip, slide,membrane, or well of a microtiter tray or plate. The arrayed nucleicacids are recombined with one or more additional nucleic acids, therebyproviding an arrayed library of recombinant nucleic acids. Theserecombinant nucleic acids are then amplified and a screened to identifymembers of the array that possess a desired property. In someembodiments, an oligonucleotide primer is tethered to the solidsubstrate and an additional single-stranded nucleic acid is annealed tothe oligonucleotide which is then extended with a nucleic acidpolymerase. In alternative embodiments, a single-stranded templatepolynucleotide is hybridized with a set of partially overlappingcomplementary nucleic acid fragments which are extended to produce anarrayed library of recombinant nucleic acids. For example, one or moretemplate nucleic acids are immobilized on a solid support. Partiallyoverlapping complementary nucleic acid fragments are annealed to thetemplate polynucleotide, and extended or ligated to produce aheteroduplex comprising the template nucleic acid and a substantiallyfull-length heterolog complementary to the template nucleic acid. Theheterolog is recovered and, optionally, further diversified. A varietyof products produced by the methods and their variants and kits andapparatus for practicing the methods are similarly described.

In the above methods, the first population of nucleic acids or thepopulation of recombinant nucleic acids are typically arranged in aphysical or logical matrix at an average of approximately 0.1-10 (e.g.,0.5-5) array members per array position. The first population of nucleicacids or the population of recombinant nucleic acids optionally includea solid phase or a liquid phase array. Optionally, the first populationof nucleic acids is provided by one or more of: synthesizing a set ofoverlapping oligonucleotides, cleaving a plurality of homologous nucleicacids to produce a set of cleaved homologous nucleic acids, step PCR ofone or more target nucleic acid, uracil incorporation and cleavageduring copying of one or more target nucleic acids, and incorporation ofa cleavable nucleic acid analogue into a target nucleic acid andcleavage of the resulting target nucleic acid. In another approach, thefirst population of nucleic acids is provided by synthesizing a set ofoverlapping oligonucleotides, by cleaving a plurality of homologousnucleic acids to produce a set of cleaved homologous nucleic acids, orboth. The set of overlapping oligonucleotides or the set of cleavedhomologous nucleic acids are optionally flowed into one or more selectedphysical locations.

The first population of nucleic acids is optionally provided bysonicating, cleaving, partially synthesizing, random primer extending ordirected primer extending one or more of: a synthetic nucleic acid, aDNA, an RNA, a DNA analogue, an RNA analogue, a genomic DNA, a cDNA, anmRNA, a DNA generated by reverse transcription, an nRNA, an aptamer, apolysome associated nucleic acid, a cloned nucleic acid, a cloned DNA, acloned RNA, a plasmid DNA, a phagemid DNA, a viral DNA, a viral RNA, aYAC DNA, a cosmid DNA, a fosmid DNA, a BAC DNA, a P1-mid, a phage DNA, asingle-stranded DNA, a double-stranded DNA, a branched DNA, a catalyticnucleic acid, an antisense nucleic acid, an in vitro amplified nucleicacid, a PCR amplified nucleic acid, an LCR amplified nucleic acid, aQ∃-replicase amplified nucleic acid, an oligonucleotide, a nucleic acidfragment, a restriction fragment and/or a combination thereof.

The first population of nucleic acids is optionally modified bypurifying one or more member of the first population of nucleic acids.Optionally, the first population of nucleic acids is provided bytransporting one or more members of the population from one or moresources of one or more members of the first population to one or moredestinations of the one or more members of the first population ofnucleic acids. For example, the transporting optionally includes flowingthe one or more members from the source to the destination. The one ormore sources of nucleic acids can include any of: a solid phase array, aliquid phase array, a container, a microtiter tray, a microtiter traywell, a microfluidic chip, a test tube, a centrifugal rotor, amicroscope slide, and/or a combination thereof.

Amplifying the recombinant nucleic acids in the physical or logicalarray of recombinant nucleic acids, or amplifying the elongated nucleicacids in the master array optionally includes one or more amplificationtechnique selected from: PCR, LCR, SDA, NASBA, TMA and Q∃-replicaseamplification. Optionally, amplifying the recombinant nucleic acids inthe physical or logical array or amplifying the elongated nucleic acidsin the master array comprises heating or cooling the physical or logicalarray or the master array, or a portion thereof.

Amplifying the recombinant nucleic acids in the physical or logicalarray or amplifying the elongated nucleic acids in the master array caninclude incorporating one or more transcription or translation controlsubsequence into one or more of: the elongated nucleic acids, therecombinant nucleic acids in the physical or logical array, anintermediate nucleic acid produced using the elongated nucleic acids orthe recombinant nucleic acids in the physical or logical array as atemplate, or a partial or complete copy of the elongated nucleic acidsor the recombinant nucleic acids in the physical or logical array. Theone or more transcription or translation control subsequence isoptionally ligated to into one or more of: the elongated nucleic acids,the recombinant nucleic acids in the physical or logical array, anintermediate nucleic acid produced using the elongated nucleic acids orthe recombinant nucleic acids in the physical or logical array as atemplate, and a partial or complete copy of the elongated nucleic acidsor the recombinant nucleic acids in the physical or logical array. Theone or more transcription or translation control subsequence isoptionally hybridized or partially hybridized to one or more of: theelongated nucleic acids, the recombinant nucleic acids in the physicalor logical array, an intermediate nucleic acid produced using theelongated nucleic acids or the recombinant nucleic acids in the physicalor logical array as a template, or a partial or complete copy of theelongated nucleic acids or the recombinant nucleic acids in the physicalor logical array.

In one aspect, the recombinant nucleic acids in the physical or logicalarray or the elongated nucleic acids in the master array are amplifiedin a DNA micro-amplifier. The micro-amplifier can include one or moreof: a programmable resistor, a micromachined zone heating chemicalamplifier, a chemical denaturation device, an electrostatic denaturationdevice, and/or a microfluidic electrical fluid resistance heatingdevice. Similarly, the physical or logical array, or portion thereof orthe master array or portion thereof, is heated or cooled by one or moreof: a Peltier solid state heat pump, a heat pump, a resistive heater, arefrigeration unit, a heat sink, and a Joule Thompson cooling device.The methods optionally include producing a duplicate amplified physicalor logical array of recombinant nucleic acids.

The methods can similarly include in vitro transcribing members of theamplified physical or logical array of recombinant nucleic acids toproduce an amplified array of in vitro transcribed nucleic acids. In oneaspect, screening the amplified physical or logical array of recombinantnucleic acids, or a duplicate thereof, for a desired property comprisesassaying a protein or product nucleic acid encoded by one or moremembers of the amplified physical or logical array of recombinantnucleic acids for one or more property.

In one aspect, the invention provides recombination of nucleic acidsusing a single-stranded template. In the methods, a first population ofsingle-stranded template polynucleotides is provided. The templatepolynucleotides are the same or different. The templates are recombinedby: (i) annealing a plurality of partially overlapping complementarynucleic acid fragments; and, (ii) extending the annealed fragments toproduce a physical or logical array comprising a first population ofrecombinant nucleic acids. In one embodiment, a physical arraycomprising the first population of template polynucleotides is providedimmobilized on a solid support (e.g., a glass support, a plasticsupport, a silicon support, a chip, a bead, a pin, a filter, a membrane,a microtiter plate, a slide or the like). In one embodiment, the firstpopulation of template polynucleotides comprises substantially an entiregenome (e.g., a bacterial or fungal genome). In another embodiment, thefirst population of template polynucleotides comprises substantially allof the expression products of a cell (e.g., eukaryotic or prokaryotic),tissue or organism. Optionally, the first population of templatepolynucleotides comprises a subset of the expression products of a cell,tissue or organism. The first population of template polynucleotidesoptionally comprises a library of genomic nucleic acids or cellularexpression products (e.g., mRNAs, cDNAs, etc.).

The template polynucleotides optionally include one or more of: a codingRNA, a coding DNA, an antisense RNA, and antisense DNA, a non-codingRNA, a non-coding DNA, an artificial RNA, an artificial DNA, a syntheticRNA, a synthetic DNA, a substituted RNA, a substituted DNA, a naturallyoccurring RNA, a naturally occurring DNA, a genomic RNA, a genomic DNA,a cDNA, or the like.

In one aspect, members of the amplified physical or logical arrays ofrecombinant nucleic acids herein are transcribed to produce an amplifiedarray of transcribed nucleic acids. These can be translated to producean amplified physical or logical array of polypeptides. Theconcentration of polypeptide or transcribed nucleic acids can bedetermined at one or more positions in the amplified physical or logicalarray of polypeptides.

In one aspect, the invention provides for re-arraying the amplifiedphysical or logical array of polypeptides or in vitro transcribednucleic acids in a secondary polypeptide or in vitro transcribed nucleicacid array which has an approximately uniform concentration ofpolypeptides or in vitro transcribed nucleic acids at a plurality oflocations in the secondary polypeptide array. Alternately, or inconjunction, a correction factor which accounts for variation inpolypeptide or in vitro transcribed nucleic acid concentrations atdifferent positions in the amplified physical or logical array ofpolypeptides or in vitro transcribed nucleic acids can be applied tonormalize detectable data.

In one aspect, one or more substrate is added to a plurality of membersof the logical array of polypeptides or in vitro transcribed nucleicacids. Formation of a product produced by contact between the one ormore substrate and one or more of the plurality of members of thelogical array of polypeptides can be monitored, directly or indirectly.Formation of the product is detected, e.g., by a coupled enzymaticreaction which detects the product or the substrate or a secondaryproduct of the product or substrate. For example, peroxide productioncan be monitored. Similarly, formation of the product is optionallydetected by monitoring production of heat or entropy which results fromthe formation of the product.

The physical or logical array of polypeptides is optionally selected fora desired property, thereby identifying one or more selected member ofthe physical or logical array of polypeptides which has a desiredproperty, and identifying one or more selected member of the amplifiedphysical or logical array of recombinant nucleic acids that encodes theone or more member of the physical or logical array of polypeptides. Forexample, the selecting is optionally performed in a primary screeningassay, comprising one or more of: (i) re-selecting the one or moreselected member of the amplified physical or logical array ofrecombinant nucleic acids in a secondary screening assay; (ii)quantifying protein levels at one or more location in the physical orlogical array of polypeptides; (iii) purifying proteins from one or morelocations in the physical or logical array of polypeptides; (iv)normalizing activity levels in the primary screen by compensating forprotein quantitation at a plurality of locations in the physical orlogical array of polypeptides; (v) determining a physical characteristicof the one or more selected members; and, (vi) determining an activityof the one or more selected members. In a further aspect, the one ormore selected member of the amplified physical or logical array ofrecombinant nucleic acids are recombined with one or more additionalnucleic acids, in vivo, in vitro or in silico.

One or more member of the amplified physical or logical array, or aduplicate thereof, can be selected based upon the screening of theamplified physical or logical array for a desired property. Optionally,a plurality of members of the amplified physical or logical array orduplicate thereof are selected, recombined and re-arrayed to form asecondary array of recombined selected nucleic acids, which secondaryarray is re-screened for the desired property, or for a second desiredproperty.

Methods of detecting or enriching for in vitro transcription ortranslation products are also provided. In the methods, one or morefirst nucleic acids which encode one or more moieties are localizedproximal to one or more moiety recognition agents which specificallybind the one or more moieties. The one or more nucleic acids are invitro translated or transcribed, producing the one or more moieties(e.g., polypeptides or biologically active RNAs such as anti-sense orribozyme molecules, or other product molecules). The one or moremoieties diffuse or flow into contact with the one or more moietyrecognition agents. Binding of the one or more moieties to the one ormore moiety recognition agents is permitted and the one or more moietiesare detected or enriched for by detecting or collecting one or morematerials proximal to, within or contiguous with the moiety recognitionagent (the material comprises at least one of the one or more moieties,where the moieties comprise one or more in vitro translation ortranscription product). Optionally, the one or more moieties are pooledby pooling the material which is collected. Here again, a variety ofvariants of this basic class of methods are set forth herein as are avariety of products produced by the methods and their variants.

Optionally, the one or more moieties (e.g., polypeptides or RNAs) arepooled by pooling the material which is collected. The moietyrecognition agents noted above optionally include one or more antibodyor one or more second nucleic acids. The first nucleic acids optionallyinclude a related population of mutated, shuffled or otherwise alterednucleic acids. In another aspect, the first nucleic acids optionallyinclude a related population of mutated, shuffled or otherwise alterednucleic acids which encode an epitope tag bound by the moiety or the oneor more moiety recognition agents.

In one aspect, the first nucleic acids comprise a related population ofmutated, shuffled or otherwise altered nucleic acids and a PCR primerbinding region. Alternately, the first nucleic acids optionally comprisea related population of mutated, shuffled or otherwise altered nucleicacids and a PCR primer binding region. In this embodiment, the methodfurther includes identifying one or more target first nucleic acid byproximity to the moieties which are bound to the one or more moietyrecognition agent, and amplifying the target first nucleic acid byhybridizing a PCR primer to the PCR primer binding region and extendingthe primer with a polymerase. The method optionally includes PCRamplifying a set of parental nucleic acids to produce the relatedpopulation of mutated, shuffled or otherwise altered nucleic acids.

In one typical embodiment, the first nucleic acids comprise an inducibleor constitutive heterologous promoter. The first nucleic acids and theone or more moiety recognition agents are typically localized on a solidsubstrate (e.g., a bead, chip, slide or the like). In one embodiment,the first nucleic acids and the one or more moiety recognition agentsare localized on the solid substrate by one or more of: a cleavablelinker chemical linker, a gel, a colloid, a magnetic field, and anelectrical field.

An activity of the moiety or moiety recognition agent is typicallydetected and the one or more first nucleic acid coupled to the moiety ormoiety recognition agent is picked with an automated robot, e.g., byplacing a capillary on a region comprising the detected activity of themoiety or moiety recognition agent. The moiety or moiety in contact withthe moiety recognition agent is optionally cleaved at a cleavable linkerwhich attaches the first nucleic acid to a solid substrate, providingfor isolation of the first nucleic acid.

Methods of producing duplicate arrays of shuffled or mutagenized nucleicacids are provided. In the methods, a physical or logical array ofshuffled or mutagenized nucleic acids or transcribed shuffled ortranscribed mutagenized nucleic acids is provided. A duplicate array ofcopies (generated, e.g., using a polymerase or nucleic acid synthesizer)of the shuffled or mutagenized nucleic acids or copies of thetranscribed shuffled or transcribed mutagenized nucleic acids is formedby physically or logically organizing the copies into a physical orlogical array. Once again, a variety of variants of this basic class ofmethods are set forth herein, as are a variety of products produced bythe methods and their variants.

In one aspect, an array of reaction mixtures which corresponds to thephysical or logical array of shuffled or mutagenized nucleic acids ortranscribed shuffled or transcribed mutagenized nucleic acids is formed.The reaction mixtures include members of the array of shuffled ormutagenized nucleic acids or transcribed shuffled or transcribedmutagenized nucleic acids or the duplicate array of copies of theshuffled or mutagenized nucleic acids or copies of the transcribedshuffled or transcribed mutagenized nucleic acids, or a derivative copythereof. The reaction mixtures typically further include one or more invitro transcription or translation reagent.

Methods of normalizing an array of reaction mixtures are provided. Inthe methods, a physical or logical array of diversified (e.g., shuffledor mutagenized) nucleic acids or transcribed shuffled or transcribedmutagenized nucleic acids is in vitro transcribed or translated toproduce an array of products. A correction factor is determined whichaccounts for variation in concentration of the products at differentsites in the array of products. Typically, a secondary product array isproduced which comprises selected concentrations of the products at oneor more sites in the secondary array, e.g., by transferring aliquotsfrom a plurality of sites in the array of products to a plurality ofsecondary sites in the secondary array. Optionally, the products arediluted while being transferred or after transfer to the secondarysites, thereby selecting the concentration of the products at thesecondary sites in the secondary array.

In one aspect, the invention provides methods of directing nucleic acidfragmentation using a computer. The method includes calculating a ratioof uracil to thymidine, which ratio when used in a fragmentation moduleproduces one or more nucleic acid fragment of a selected length.

In another aspect, methods of directing PCR using a computer areprovided. The method includes calculating one or more crossover regionbetween two or more parental nucleic acid sequence using one or moreannealing temperature or extension temperature. For example, the methodoptionally includes calculating the one or more crossover region usingone or more theoretical prediction or one or more set of empirical datato calculate a melting temperature.

Methods of selecting one or more parental nucleic acids for diversitygeneration using a computer are also provided. In the method, analignment between two or more potential parental nucleic acid sequencesis performed. A number of mismatches between the aligned sequences iscalculated and a melting temperature for one or more window of w basesin the alignment is calculated. One or more window of w bases having amelting temperature greater than x is determined and one or morecrossover segment in the alignment is identified, which one or morecrossover segment comprises two or more windows having a meltingtemperature greater than x, which two or more windows are separated byno more than n nucleotides. A dispersion of the one or more crossoversegments is calculated and a first score for each alignment based on thenumber of windows having a melting temperature greater that x, thedispersion, and the number of crossover segments identified iscalculated. A second score based on the number of mismatches, the numberof windows having a melting temperature greater that x, the dispersion,and the number of crossover segments identified is determined, and oneor more parental nucleic acid is selected based on the first scoreand/or the second score. These steps are optionally repeated, e.g.,starting with the one or more parental nucleic acid which are selected.

In this method, the alignment optionally comprises a pairwise alignment.W optionally comprises an odd number, e.g., about 21. The methodoptionally includes calculating the melting temperature for the one ormore window of w bases in the alignment from one or more set ofempirical data or one or more melting temperature prediction algorithm.Example values for x include about 65° C. Example values for n includeabout 2. In the methods, the dispersion typically comprises the inverseof the average number of bases between crossover segments in thealignment.

Typically, the instruction set selects the two or more potentialparental nucleic acid sequences by searching one or more database forone or more nucleic acid sequence of interest and one or more homolog ofthe one or more nucleic acid sequence of interest.

The invention further provides embodiments in a web page, e.g., fordirecting nucleic acid diversity generation, the web page comprising acomputer readable medium that causes a computer to perform any of themethods herein.

Products produced by any of the processes herein are a feature of theinvention.

Kits embodying the methods and comprising various components of thedevice/apparatus/integrated systems herein are also provided. Use of themethods and/or device/systems for any of the purposes indicated hereinare also a feature of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1, Panels A and B is a schematic flow chart of an integrated systemof the invention, beginning with input nucleic acids.

FIG. 2 provides an example schematic of the modules of an integratedshuffling machine.

FIG. 3 provides a schematic representation of the steps performed by anexemplar shuffling module. As shown, a single pot reaction is performed,utilizing uracil incorporation, DNA fragmentation and assembly. A rescuePCR is performed, the results assessed with PicoGreen and any wells thattest positive for PicoGreen incorporation are rescued and sent to thelibrary quality modules.

FIG. 4 provides a schematic overview of an exemplar Library QualityModule.

FIG. 5 provides a schematic overview of an exemplar dilution module'sactivities.

FIG. 6 provides a schematic overview of the activities of an exemplarexpression module.

FIG. 7 provides a schematic overview of the activities of an exemplarassay module.

FIG. 8 is a schematic of an example recombination and selection machine.

FIG. 9, panels A-B provide a schematic illustration of various detectionstrategies using single or multiple primers (e.g., via TaqMan).

FIG. 10 is a schematic of an example DNA shuffling machine.

FIG. 11 is a schematic of a DNA fragmentation device or module.

FIG. 12 is a schematic of a DNA fragment analysis and isolation deviceor module.

FIG. 13 is a schematic of a DNA fragment prep device.

FIG. 14 is a schematic of a precision microamplifier.

FIG. 15 is a schematic of a DNA assembly and rescue module.

FIG. 16 is a schematic of a recombination analysis module.

FIG. 17, panels A-E is a schematic of exemplar enrichment methods for invitro transcription/translation.

FIG. 18 is a schematic of a high-throughput parallel SPR module.

FIG. 19 is a schematic of a shuffling chip.

FIG. 20 is a schematic of the fluidics layer of a shuffling system.

FIG. 21 is a schematic of an environmental control layer.

FIG. 22 is a schematic of a microscale appliance.

FIG. 23 is a schematic outline of processes for sourcing nucleic acidsfrom diverse sources.

FIG. 24 is an alternative schematic outline of processes for sourcingnucleic acids from diverse sources.

FIG. 25 is an alternative schematic outline of processes for sourcingnucleic acids from diverse sources.

FIG. 26 is an alternative schematic outline of processes for sourcingnucleic acids from diverse sources.

FIG. 27 is an alternative schematic outline of processes for sourcingnucleic acids from diverse sources.

FIG. 28 is an alternative schematic outline of processes for sourcingnucleic acids from diverse sources.

FIG. 29 is an alternative schematic outline of processes for sourcingnucleic acids from diverse sources.

FIG. 30 is an alternative schematic outline of processes for sourcingnucleic acids from diverse sources.

FIG. 31 schematically illustrates recombination of nucleic acidstethered to a solid support.

FIGS. 32A and B schematically illustrate recovery procedures using“boomerang” and “vectorette” amplification strategies.

FIG. 33 is an illustration of the melting temperature for a nucleic acidpairwise hybridization showing various crossover segments.

I. DEFINITIONS

The following definitions supplement those common in the art for theterms specified.

A “physical array” is a set of specified elements arranged in aspecified or specifiable spatial arrangement. A “logical array” is a setof specified elements arranged in a manner which permits access to theelements of the set. A logical array can be, e.g., a virtual arrangementof the set in a computer system, or, e.g., an arrangement of setelements produced by performing a specified physical manipulation on oneor more set element or components of set elements. For example, alogical array can be described in which set elements (or components thatcan be combined to produce set elements) can be transported ormanipulated to produce the set. A “duplicate” or “copy” array is anarray which can be at least partially corresponded to a parental array.In simplest form, this correspondence takes the form of simplyreplicating all or part of the parental array, e.g., by taking analiquot of material from each position in the parental array and placingthe aliquot in a defined position in the duplicate array. However, anymethod which results in the ability to correspond members of theduplicate array to the parental array can be used for array duplication,including the use of simple or complex storage algorithms, partially orpurely in silico arrays, and pooling approaches which partially combinesome elements of the parental array into single locations (physical orvirtual) in the duplicate array. The duplicate or copy array duplicatessome or all components of a parental array. For example, an array ofreaction mixtures optionally includes nucleic acids and translation ortranscription reagents at sites in the array, while the duplicate/copyarray can also include the complete reaction mixtures, or, alternately,can include, e.g., the nucleic acids, without the other reaction mixturecomponents.

A “shuffled” nucleic acid is a nucleic acid produced by a shufflingprocedure such as any shuffling procedure set forth herein. Shufflednucleic acids are produced by recombining (physically or virtually) twoor more nucleic acids (or character strings), e.g., in an artificial,and optionally recursive, fashion. Generally, one or more screeningsteps are used in shuffling processes to identify nucleic acids ofinterest; this screening step can performed before or after anyrecombination step. In some (but not all) shuffling embodiments, it isdesirable to perform multiple rounds of recombination prior to selectionto increase the diversity of the pool to be screened. The overallprocess of recombination and selection are optionally repeatedrecursively. Depending on context, shuffling can refer to an overallprocess of recombination and selection, or, alternately, can simplyrefer to the recombinational portions of the overall process.

A “mutagenized nucleic acid” is a nucleic acid which has been physicallyaltered as compared to a parental nucleic acid (e.g., such as anaturally occurring nucleic acid), e.g., by modifying, deleting,rearranging, or replacing one or more nucleotide residue in themutagenized nucleic acid as compared to the parental nucleic acid.

A “transcribed” nucleic acid is a nucleic acid produced by copying aparental nucleic acid, where the parental nucleic acid is a differentnucleic acid type than the copied nucleic acid. For example, an RNA copyof a DNA molecule (e.g., as occurs during classical transcription) or aDNA copy of an RNA molecule (e.g., as occurs during classical reversetranscription) can be a “transcribed nucleic acid” as that term isintended herein. Similarly, artificial nucleic acids, including peptidenucleic acids, can be used as either the parental or the copied nucleicacid (and artificial nucleotides can be incorporated into eitherparental or copied molecules). Copying can be performed, e.g., usingappropriate polymerases, or using in vitro artificial chemical syntheticmethods, or a combination of synthetic and enzymatic methods.

An “in vitro translation reagent” is a reagent which is necessary orsufficient for in vitro translation, or a reagent which modulates therate or extent of an in vitro translation reaction, or which alters theparameters under which the reaction is operative. Examples includeribosomes, and reagents which include ribosomes, such as reticulocytelysates, bacterial cell lysates, cellular fractions thereof, aminoacids, t-RNAs, etc.

A “translation product” is a product (typically a polypeptide) producedas a result of the translation of a nucleic acid. A “transcriptionproduct” is a product (e.g., an RNA, optionally including mRNA, or,e.g., a catalytic or biologically active RNA) produced as a result oftranscription of a nucleic acid.

A “solid phase array” is an array in which the members of the array arefixed to or within a solid or semi-solid substrate. The fixation can bethe result of any interaction that tends to immobilize components,including chemical linking, heat treatment, hybridization,ligand/receptor interactions, metal chelation interactions, ionexchange, hydrogen bonding and hydrophobic interactions and the like.For semi-solid substrates such as gels and gel droplets, linking mayrequire nothing more than mixing of the member with the substratematerial during or after solidification. A “solid substrate” has a fixedorganizational support matrix, such as silica, glass, polymericmaterials, membranes, filters, beads, pins, slides, microtiter plates ortrays, etc. In some embodiments, at least one surface of the substrateis partially planar, but in others, the solid substrate is a discreteelement such as a bead which can be dispensed into an organizationmatrix such as a microtiter tray. Solid support materials include, butare not limited to, glass, polacryloylmorpholide, silica, controlledpore glass (CPG), polystyrene, polystyrene/latex, polyacyrlate,polyacrylamide, agar, agarose, chemically modified agars and agaroses,carboxyl modified teflon, nylon and nitrocellulose. The solid substratescan be biological, nonbiological, organic, inorganic, or a combinationof any of these, existing as particles, strands, precipitates, gels,sheets, tubing, spheres, containers, capillaries, pads, slices, films,plates, slides, etc., depending upon the particular application. Othersuitable solid substrate materials will be readily apparent to those ofskill in the art. Often, the surface of the solid substrate will containreactive groups, such as carboxyl, amino, hydroxyl, thiol, or the likefor the attachment of nucleic acids, proteins, etc. Surfaces on thesolid substrate will sometimes, though not always, be composed of thesame material as the substrate. Thus, the surface may be composed of anyof a wide variety of materials, for example, polymers, plastics, resins,polysaccharides, silica or silica-based materials, carbon, metals,inorganic glasses, membranes, or any of the above-listed substratematerials. The surface may also be chemically modified or functionalizedin such a way as to allow it to establish binding interactions withfunctional groups intrinsic to or specifically associated with thenucleic acids or polypeptides to be immobilized.

A “liquid phase array” is an array in which the members of the array arefree in solution, e.g., on a microtiter tray, or in a series ofcontainers such as a set of test tubes or other containers. Most often,members of a liquid phase array are separated in space by subdividingthe volume containing the members of the array into multiple discretechambers such that each chamber contains less than a complete library ofmembers, and ideally less than about 10% of the discrete members in thelibrary. Such separation or fractionation of a population containing aplurality of unique sequences can be accomplished by sorting, dilution,serial dilution, and a variety of other methods.

Nucleic acids are “homologous” when they derive (artificially ornaturally) from a common ancestor. Where there is no direct knowledge ofthe relatedness of two or more nucleic acids, homology is often inferredby consideration of the percent identity or by identification ofdiscrete sequence motifs within sets of low identity sequences of therelevant nucleic acids. As described in more detail herein, commonlyavailable software programs such as BLAST and PILEUP can be used tocalculate relatedness of nucleic acids.

Nucleic acids “hybridize” when they preferentially associate insolution. As described in more detail below, a variety of parameterssuch as temperature, ionic buffer conditions and the presence or absenceof organic solvents affect hybridization of two or more nucleic acids.

A “translation control sequence” is a nucleic acid subsequence whichaffects the initiation, rate or extent of translation of a nucleic acid,such as ribosome binding sites, stop codons and the like. A variety ofsuch sequences are known and described in the references set forthherein and many more are fully available to one of skill.

A “transcription control sequence” is a nucleic acid subsequence whichaffects the initiation, rate or extent of transcription of a nucleicacid, such as a promoter, enhancer or terminator sequences. A variety ofsuch sequences are known and described in the references set forthherein, and many more are fully available to one of skill.

DETAILED DISCUSSION OF THE INVENTION

The present invention takes advantage of a variety of technologies toautomate nucleic acid shuffling and other diversity-generation dependentprocesses. Each aspect of diversity generation and downstream screeningprocesses can be automated (and used individually in separate modules orcollectively in an integrated system or an overall device), providingdevices, systems and methods which greatly increase throughput forgenerating diverse nucleic acids (e.g., by recombination methods such asDNA shuffling, or via other mutagenesis methods, or combinationsthereof) and screening for desirable properties of those nucleic acids(e.g., encoded RNAs, proteins, or the like).

The invention provides, among other things, methods, kits, devices andintegrated systems. For example, devices and integrated systemscomprising a physical or logical array of reaction mixtures areprovided. Each reaction mixture comprises one or more recombinant,shuffled or otherwise diversified nucleic acids (e.g., diversified bymutagenesis, optionally including recombination or other methods), orcorresponding transcribed nucleic acids (e.g., cDNAs or mRNAs). Thereaction mixtures of the array also include one or more in vitrotranscription and/or translation reagents.

As will be described in more detail below, arrays can be, and commonlyare, partially or completely duplicated in the methods and systems ofthe invention. For example, aliquots of reaction mixtures or productscan be taken and copy arrays formed from the aliquots. Similarly, masterarrays comprising, e.g., the nucleic acids found in the reactionmixtures (e.g., arrays constituted of duplicate amplified sets ofdiversified nucleic acids) can be produced. The precise manner ofproduction of array copies varies according to the physical nature ofthe array. For example, where arrays are formed in microtiter trays,copy arrays are conveniently formed in microtiter trays, e.g., byautomated pipetting of aliquots of material from an original array.However, arrays can also change form in the copying process, i.e.,liquid phase copies can be formed from solid phase arrays, or viceversa, or a logical array can be converted to a simple or complexspatial array in the process of forming the copy (e.g., by moving orcreating an aliquot of material corresponding to a member of the logicalarray, and, subsequently, placing the aliquot with other array membersin an accessible spatial relationship such as a gridded array), or viceversa (e.g., array member positions can be recorded and that informationused as the basis for logical arrays that constitute members of multiplespatial arrays—a common process when identifying “hits” having anactivity of interest).

The arrays can include both reaction mixture and product components. Forexample, in addition to the nucleic acids, transcription regents andtranslation reagents noted above, the arrays can also include productsof the reaction mixture such as RNAs (e.g., mRNAs, biologically activenucleic acids (e.g., ribozymes, aptamers, antisense molecules, etc.)proteins, or the like. Thus, the reaction mixtures can comprise one ormore translation products or one or more transcription products, orboth.

Similarly, the arrays can have any of a variety of physicalconfigurations, including solid or liquid phase(s). Some or all of thecomponents of the reaction mixtures can be fixed in position, e.g., thenucleic acids in the reaction mixtures can be relatively fixed inposition (e.g., in a solid or immobilized phase), while the othercomponents of the array can diffuse across the array (e.g., through agel or other immobilizing matrix). Alternatively, some or all of themembers of the array can be immobilized to a single general spatiallocation (e.g., by being present in wells of a microtiter dish, eitherby being fixed to the surface of the dish or in solution in the wells ofthe dish). Thus, the array of reaction mixtures can comprises a solidphase or a liquid phase array of any of the components of the reactionmixtures, e.g., the diversified nucleic acids (or transcribed productsthereof), in vitro translation reagents, etc.

I. An Overview of Integrated Diversity Generation/Screening Systems

FIG. 1, panels A and B provides a schematic overview of an exampleintegrated system of the invention. In some contexts, some of the listedelements are omitted; conversely, many additional elements areoptionally included.

As shown, nucleic acids (DNA, RNA, etc.) or corresponding characterstrings (e.g., characters in a computer system) are input into thesystem. A diversity generation module (e.g., a shuffling and/ormutagenesis module) recombines, mutagenizes or otherwise modifies theinput nucleic acids to produce a diverse set of nucleic acids that areused to produce one or more product (a protein, bioactive RNA, or thelike) in a product production module. Variant nucleic acids are thenselected (typically by screening products from the production module)for a desired encoded activity (encoded protein or RNA, level of RNAexpression, level of protein expression, etc.). Top variants are thenselected for further characterization, additional rounds of diversitygeneration (e.g., recombination of the top variants with each other orwith additional nucleic acids, or both).

Typically, a product quantification module can be used to normalizeselection results (i.e., to account for differences in concentrations ofprotein, catalytic RNAs or other products). Optionally, one or moreadditional secondary assay can be performed to further select for one ormore additional property of interest in any product.

FIG. 1, panel B provides additional details of the example integratedsystem. As shown, nucleic acids are dispensed from diversity generationmodule 1 into microtiter trays (as described below, many alternativeconfigurations that do not use such trays, instead using other liquid(e.g., microfluidic) or solid phase arrays). For example, thediversified DNAs (or other nucleic acids) are dispensed into first trayor set of trays 10 at about 0-100 unique DNA molecules/well to providefor straightforward interpretation of results from the system. Commonly,each well can contain 0-10 unique molecules. For example, each well cancontain, on average, 0-5, or e.g., 0-3 unique molecules. That is, ifthere are only 1 or a few nucleic acid molecule member types per arrayposition it is easier to identify which array members produce adesirable activity. However, arrays of pooled members can be used, inwhich pools having an activity of interest are subsequently deconvoluted(e.g., re-arrayed by limiting dilution and the pool members tested forany activity of interest). In this context, the term “unique” refers tonucleic acids of differing lengths or sequences.

A nucleic acid master array is produced by amplifying the members of thefirst tray (the amplified members are accessible for furtheroperations), e.g., as indicated by PCR process amplification step(s) 15.One or more copies of this master array (20, 21) is optionally produced(e.g., by aliquotting or otherwise transferring materials from theoriginal to the copies) for further access by the system in subsequentprocedures. Either the original or the duplicate of the master array canbe in vitro transcribed (if appropriate—the copying procedure(represented by in vitro transcription process step 25) can produce DNAor RNA copies (e.g., as represented by mRNA copy array 30), and theoriginal can be DNA or RNA, as desired) and/or translated in vitro toproduce a product of interest (e.g., a biologically active RNA, protein,or the like, represented by protein/RNA array 40). This is representedby in vitro transcription process step(s) 35.

The product is assayed as appropriate on primary assay plate 50 whichoptionally includes substrates or other relevant components. Secondaryassays (i.e., assays for activities which differ from the firstactivity) can also be run in secondary assay modules.

Typically, a product quantification module such as a proteinquantification/purification module 60 is used to normalize the activitylevel of the product, i.e., to detect and/or account for variation inproduct concentrations. Protein quantitation module 60 allows arrayingat uniform concentration for specific activities. Aliquots of existingproteins can be rearrayed and reassayed, e.g., on secondary assay plate70. New protein can be reproduced from mRNA or dsDNA, quantified andreassayed.

Detector elements are typically included in protein quantitation module60 to detect product activities of interest (hits). Optionally, hitpicking software and or hardware is used to select hits (other softwareelements control sample manipulation and transfer between modules andrespond to user inputs). The system determines which nucleic acids inthe master array that the hits correspond to and either identifies thehits to the user or uses corresponding nucleic acids from the originalor copy master array in subsequent diversity generation reactions, suchas in additional shuffling reactions in the diversity generation module.

In general in FIG. 1, arrows between plates indicate processes that canbe used to produce new plates, or which can be performed on existingplates.

II. Methods and System Elements for Generating Nucleic Acid Diversity

A variety of diversity generating protocols (e.g., mutation, includingrecombination and other methods) are available and described in the art.The procedures can be used separately, and/or in combination to produceone or more variants of a nucleic acid or set of nucleic acids, as wellvariants of encoded proteins. Individually and collectively, theseprocedures provide robust, widely applicable ways of generatingdiversified nucleic acids and sets of nucleic acids (including, e.g.,nucleic acid libraries) useful, e.g., for the engineering or rapidevolution of nucleic acids, proteins, pathways, cells and/or organismswith new and/or improved characteristics.

While distinctions and classifications are made in the course of theensuing discussion for clarity, it will be appreciated that thetechniques are often not mutually exclusive. Indeed, the various methodscan be used singly or in combination, in parallel or in series, toprovide diverse sequence variants.

The result of any of the diversity generating procedures describedherein can be the generation of one or more nucleic acids, which can beselected or screened for nucleic acids that encode proteins or bioactiveRNAs (e.g., catalytic RNAs) with or which confer new or desirableproperties. Following diversification by one or more of the methodsherein, or otherwise available to one of skill, any nucleic acids thatare produced can be selected for a desired activity or property, e.g.for use in the automated systems and methods herein. This can includeidentifying any activity that can be detected, for example, in anautomated or automatable format, by any of the assays in the art orherein. A variety of related (or even unrelated) properties can beevaluated, in serial or in parallel, at the discretion of thepractitioner.

As noted, a variety of diversity generating/product screening reactionscan be automated by the methods set forth herein. One important class ofsuch reactions are “nucleic acid shuffling” or “DNA shuffling” methods.In these methods, any of a variety of recombination-based diversitygenerating procedures can be used to diversify starting nucleic acids,or organisms comprising nucleic acids, or even to diversify characterstrings which are “in silico” (in computer) representations of nucleicacids (or both). Diverse nucleic acids/character strings/organisms whichare generated by such methods are typically screened for one or moreactivity. Nucleic acids, character strings, or organisms which comprisenucleic acids are then optionally used as substrates in subsequentrecombination reactions, the products of which are, again, screened forone or more activity. This process is optionally repeated recursivelyuntil one or more desirable product is produced.

A variety of diversity generating protocols, including nucleic acidshuffling protocols, are available and fully described in the art. Thefollowing publications describe a variety of recursive recombination andother mutational procedures and/or methods which can be incorporatedinto such procedures, as well as other diversity generating protocols:Soong, N. et al. (2000) “Molecular breeding of viruses” Nat Genet.25(4):436-439; Stemmer, et al., (1999) “Molecular breeding of virusesfor targeting and other clinical properties. Tumor Targeting” 4:1-4;Nesset al. (1999) “DNA Shuffling of subgenomic sequences of subtilisin”Nature Biotechnology 17:893-896; Chang et al. (1999) “Evolution of acytokine using DNA family shuffling” Nature Biotechnology 17:793-797;Minshull and Stemmer (1999) “Protein evolution by molecular breeding”Current Opinion in Chemical Biology 3:284-290; Christians et al. (1999)“Directed evolution of thymidine kinase for AZT phosphorylation usingDNA family shuffling” Nature Biotechnology 17:259-264; Crameri et al.(1998) “DNA shuffling of a family of genes from diverse speciesaccelerates directed evolution” Nature 391:288-291; Crameri et al.(1997) “Molecular evolution of an arsenate detoxification pathway by DNAshuffling,” Nature Biotechnology 15:436-438; Zhang et al. (1997)“Directed evolution of an effective fucosidase from a galactosidase byDNA shuffling and screening” Proceedings of the National Academy ofSciences U.S.A. 94:4504-4509; Patten et al. (1997) “Applications of DNAShuffling to Pharmaceuticals and Vaccines” Current Opinion inBiotechnology 8:724-733; Crameri et al. (1996) “Construction andevolution of antibody-phage libraries by DNA shuffling” Nature Medicine2:100-103; Crameri et al. (1996) “Improved green fluorescent protein bymolecular evolution using DNA shuffling” Nature Biotechnology14:315-319; Gates et al. (1996) “Affinity selective isolation of ligandsfrom peptide libraries through display on a lac repressor ‘headpiecedimer’” Journal of Molecular Biology 255:373-386; Stemmer (1996) “SexualPCR and Assembly PCR” In: The Encyclopedia of Molecular Biology. VCHPublishers, New York. pp. 447-457; Crameri and Stemmer (1995)“Combinatorial multiple cassette mutagenesis creates all thepermutations of mutant and wildtype cassettes” BioTechniques 18:194-195;Stemmer et al., (1995) “Single-step assembly of a gene and entireplasmid form large numbers of oligodeoxyribonucleotides” Gene,164:49-53; Stemmer (1995) “The Evolution of Molecular Computation”Science 270: 1510; Stemmer (1995) “Searching Sequence Space”Bio/Technology 13:549-553; Stemmer (1994) “Rapid evolution of a proteinin vitro by DNA shuffling” Nature 370:389-391; and Stemmer (1994) “DNAshuffling by random fragmentation and reassembly: In vitro recombinationfor molecular evolution.” Proceedings of the National Academy ofSciences, U.S.A. 91:10747-10751.

Additional available mutational methods of generating diversity include,for example, site-directed mutagenesis (Ling et al. (1997) “Approachesto DNA mutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale etal. (1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller & Smith (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller & Smith (1987)“Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Tayloret al. (1985) “The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764;Taylor et al. (1985) “The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA” Nucl.Acids Res. 13: 8765-8787 (1985); Nakamaye & Eckstein (1986) “Inhibitionof restriction endonuclease Nci I cleavage by phosphorothioate groupsand its application to oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 14: 9679-9698; Sayers et al. (1988) “Y-T Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 16:791-802; and Sayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350-367; Kramer et al. (1988) “Improvedenzymatic in vitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz et al. (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999).

Additional suitable methods include point mismatch repair (Kramer et al.(1984) “Point Mismatch Repair” Cell 38:879-887), mutagenesis usingrepair-deficient host strains (Carter et al. (1985) “Improvedoligonucleotide site-directed mutagenesis using M13 vectors” Nucl. AcidsRes. 13: 4431-4443; and Carter (1987) “Improved oligonucleotide-directedmutagenesis using M13 vectors” Methods in Enzymol. 154: 382-403),deletion mutagenesis (Eghtedarzadeh & Henikoff (1986) “Use ofoligonucleotides to generate large deletions” Nucl. Acids Res. 14:5115), restriction-selection and restriction-purification (Wells et al.(1986) “Importance of hydrogen-bond formation in stabilizing thetransition state of subtilisin” Phil. Trans. R. Soc. Lond. A 317:415-423), mutagenesis by total gene synthesis (Nambiar et al. (1984)“Total synthesis and cloning of a gene coding for the ribonuclease Sprotein” Science 223: 1299-1301; Sakamar and Khorana (1988) “Totalsynthesis and expression of a gene for the a-subunit of bovine rod outersegment guanine nucleotide-binding protein (transducin)” Nucl. AcidsRes. 14: 6361-6372; Wells et al. (1985) “Cassette mutagenesis: anefficient method for generation of multiple mutations at defined sites”Gene 34:315-323; and Grundström et al. (1985) “Oligonucleotide-directedmutagenesis by microscale ‘shot-gun’ gene synthesis” Nucl. Acids Res.13: 3305-3316), double-strand break repair (Mandecki (1986)“Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis” Proc. Natl.Acad. Sci. USA, 83:7177-7181; and Arnold (1993) “Protein engineering forunusual environments” Current Opinion in Biotechnology 4:450-455).Additional details on many of the above methods can be found in Methodsin Enzymology Volume 154, which also describes useful controls fortrouble-shooting problems with various mutagenesis methods.

Additional details regarding DNA shuffling and other diversitygenerating methods are found in U.S. patents by the inventors and theirco-workers, including: U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25,1997), “METHODS FOR IN VITRO RECOMBINATION;” U.S. Pat. No. 5,811,238 toStemmer et al. (Sep. 22, 1998) “METHODS FOR GENERATING POLYNUCLEOTIDESHAVING DESIRED CHARACTERISTICS BY ITERATIVE SELECTION ANDRECOMBINATION;” U.S. Pat. No. 5,830,721 to Stemmer et al. (Nov. 3,1998), “DNA MUTAGENESIS BY RANDOM FRAGMENTATION AND REASSEMBLY;” U.S.Pat. No. 5,834,252 to Stemmer, et al. (Nov. 10, 1998) “END-COMPLEMENTARYPOLYMERASE REACTION,” and U.S. Pat. No. 5,837,458 to Minshull, et al.(Nov. 17, 1998), “METHODS AND COMPOSITIONS FOR CELLULAR AND METABOLICENGINEERING.”

In addition, details and formats for recursive recombination, e.g., DNAshuffling and other diversity generating protocols are found in avariety of PCT and foreign patent application publications, including:Stemmer and Crameri, “DNA MUTAGENESIS BY RANDOM FRAGMENTATION ANDREASEMBLY” WO 95/22625; Stemmer and Lipschutz “END COMPLEMENTARYPOLYMERASE CHAIN REACTION” WO 96/33207; Stemmer and Crameri “METHODS FORGENERATING POLYNUCLEOTIDES HAVING DESIRED CHARACTERISTICS BY ITERATIVESELECTION AND RECOMBINATION” WO 97/0078; Minshul and Stemmer, “METHODSAND COMPOSITIONS FOR CELLULAR AND METABOLIC ENGINEERING” WO 97/35966;Punnonen et al. “TARGETING OF GENETIC VACCINE VECTORS” WO 99/41402;Punnonen et al. “ANTIGEN LIBRARY IMMUNIZATION” WO 99/41383; Punnonen etal. “GENETIC VACCINE VECTOR ENGINEERING” WO 99/41369; Punnonen et al.OPTIMIZATION OF IMMUNOMODULATORY PROPERTIES OF GENETIC VACCINES WO9941368; Stemmer and Crameri, “DNA MUTAGENESIS BY RANDOM FRAGMENTATIONAND REASSEMBLY” EP 0934999; Stemmer “EVOLVING CELLULAR DNA UPTAKE BYRECURSIVE SEQUENCE RECOMBINATION” EP 0932670; Stemmer et al.,“MODIFICATION OF VIRUS TROPISM AND HOST RANGE BY VIRAL GENOME SHUFFLING”WO 9923107; Apt et al., “HUMAN PAPILLOMAVIRUS VECTORS” WO 9921979; DelCardayre et al. “EVOLUTION OF WHOLE CELLS AND ORGANISMS BY RECURSIVESEQUENCE RECOMBINATION” WO 9831837; Patten and Stemmer, “METHODS ANDCOMPOSITIONS FOR POLYPEPTIDE ENGINEERING” WO 9827230; Stemmer et al.,and “METHODS FOR OPTIMIZATION OF GENE THERAPY BY RECURSIVE SEQUENCESHUFFLING AND SELECTION” WO9813487.

Certain U.S. applications provide additional details regarding variousdiversity generating methods, including “SHUFFLING OF CODON ALTEREDGENES” by Patten et al. filed Sep. 28, 1999, (U.S. Ser. No. 09/407,800);“EVOLUTION OF WHOLE CELLS AND ORGANISMS BY RECURSIVE SEQUENCERECOMBINATION”, by del Cardayre et al. filed Jul. 15, 1998 (U.S. Ser.No. 09/166,188), and Jul. 15, 1999 (U.S. Ser. No. 09/354,922);“OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION” by Crameri et al.,filed Sep. 28, 1999 (U.S. Ser. No. 09/408,392), and “OLIGONUCLEOTIDEMEDIATED NUCLEIC ACID RECOMBINATION” by Crameri et al., filed Jan. 18,2000 (PCT/US00/01203); “USE OF CODON-VARIED OLIGONUCLEOTIDE SYNTHESISFOR SYNTHETIC SHUFFLING” by Welch et al., filed Sep. 28, 1999 (U.S. Ser.No. 09/408,393); “METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES& POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” by Selifonov et al.,filed Jan. 18, 2000, (PCT/US00/01202) and, e.g., “METHODS FOR MAKINGCHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIREDCHARACTERISTICS” by Selifonov et al., filed Jul. 18, 2000 (U.S. Ser. No.09/618,579); “METHODS OF POPULATING DATA STRUCTURES FOR USE INEVOLUTIONARY SIMULATIONS” by Selifonov and Stemmer, filed Jan. 18, 2000(PCT/US00/01138); and “SINGLE-STRANDED NUCLEIC ACID TEMPLATE-MEDIATEDRECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION” by Affholter, filedSep. 6, 2000 (U.S. Ser. No. 09/656,549).

As review of the foregoing publications, patents, published applicationsand U.S. patent applications reveals, recursive recombination and othermutation methods for modifying nucleic acids to provide new nucleicacids with desired (e.g., new or improved) properties can be carried outby a number of established methods and these procedures can be combinedwith any of a variety of other diversity generating methods. Thefollowing exemplify some of the different formats for diversitygeneration in the context of the present invention, including, e.g.,certain recombination based diversity generation formats. Manyadditional formats are provided in the references above and herein, andcan be adapted to use in the systems and methods herein.

For example, several different general classes of recombination methodsare applicable to the present invention and set forth in the referencesabove. First, nucleic acids can be recombined in vitro by any of avariety of techniques discussed in the references above, including e.g.,DNAse digestion of nucleic acids to be recombined followed by ligationand/or PCR reassembly of the nucleic acids. Second, nucleic acids can berecursively recombined in vivo, e.g., by allowing recombination to occurbetween nucleic acids in cells. Third, whole genome recombinationmethods can be used in which whole genomes of cells or other organismsare recombined, optionally including spiking of the genomicrecombination mixtures with desired library components. Fourth,synthetic recombination methods can be used, in which oligonucleotidescorresponding to targets of interest are synthesized and reassembled inPCR or ligation reactions which include oligonucleotides whichcorrespond to more than one parental nucleic acid, thereby generatingnew recombined nucleic acids. Oligonucleotides can be made by standard,single nucleotide addition methods, or by methods in whichdinucleotides, trinucleotides or longer oligomers are added in at leastone synthetic cycle, for example, to limit or expand the number ofcodons which may be present at a given position within a synthetic orsemi-synthetic gene. Moreover, recombined nucleic acids may be generatedeither from a starting pool of single stranded oligonucleotides or byfirst annealing at least one single-stranded oligomer to a complementsequence, thus forming a starting pool of preannealed double strandedoligonucleotides. Fifth, in silico methods of recombination can beeffected in which genetic algorithms are used in a computer to recombinesequence strings which correspond to nucleic acid homologues (or evennon-homologous sequences). The resulting recombined sequence strings areoptionally converted into nucleic acids by synthesis of nucleic acidswhich correspond to the recombined sequences, e.g., in concert witholigonucleotide synthesis/gene reassembly techniques. Sixth, methods ofaccessing natural diversity, e.g., by hybridization of diverse nucleicacids or nucleic acid fragments to single-stranded templates, followedby polymerization and/or ligation to regenerate full-length sequences,optionally followed by degradation of the templates and recovery of theresulting modified nucleic acids can be used. Any of the precedinggeneral recombination formats can be practiced in a reiterative fashionto generate a more diverse set of recombinant nucleic acids.

Thus, as noted, nucleic acids can be recombined in vitro by any of avariety of techniques discussed in the references above, including e.g.,DNAse digestion of nucleic acids to be recombined followed by ligationand/or PCR reassembly of the nucleic acids. For example, sexual PCRmutagenesis can be used in which random (or pseudo random, or evennon-random) fragmentation of the DNA molecule is followed byrecombination, based on sequence similarity, between DNA molecules withdifferent but related DNA sequences, in vitro, followed by fixation ofthe crossover by extension in a polymerase chain reaction. This processand many process variants are described in several of the referencesabove, e.g., in Stemmer (1994) Proc. Natl. Acad. Sci. USA91:10747-10751. The present invention provides various automated formatsand related devices for practicing such methods.

Similarly, nucleic acids can be recursively recombined in vivo, e.g., byallowing recombination to occur between nucleic acids in cells. Manysuch in vivo recombination formats are set forth in the references notedabove. Such formats optionally provide direct recombination betweennucleic acids of interest, or provide recombination between vectors,viruses, plasmids, etc., comprising the nucleic acids of interest, aswell as other formats. Details regarding such procedures are found inthe references noted above. Here again, the present invention providesvarious automated formats and related devices for practicing suchmethods.

In addition, whole genome recombination methods can also be used inwhich whole genomes of cells or other organisms are recombined,optionally including spiking of the genomic recombination mixtures withdesired library components (e.g., genes corresponding to the pathways ofthe present invention). These methods have many applications, includingthose in which the identity of a target gene is not known. Details onsuch methods are found, e.g., in WO 98/31837 by del Cardayre et al.“Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” and in, e.g., PCT/US99/15972 by del Cardayre et al.,also entitled “Evolution of Whole Cells and Organisms by RecursiveSequence Recombination.” The present invention provides variousautomated formats and related devices for practicing such methods.

As noted, synthetic recombination methods can also be used, in whicholigonucleotides corresponding to targets of interest are synthesizedand reassembled in PCR or ligation reactions which includeoligonucleotides which correspond to more than one parental nucleicacid, thereby generating new recombined nucleic acids. Oligonucleotidescan be made by standard nucleotide addition methods, or can be made,e.g., by tri-nucleotide or other synthetic approaches. Details regardingsuch approaches are found in the references noted above, including,e.g., “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION” by Crameriet al., filed Sep. 28, 1999 (U.S. Ser. No. 09/408,392), and“OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION” by Crameri et al.,filed Jan. 18, 2000 (PCT/US00/01203); “USE OF CODON-VARIEDOLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” by Welch et al.,filed Sep. 28, 1999 (U.S. Ser. No. 09/408,393); “METHODS FOR MAKINGCHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIREDCHARACTERISTICS” by Selifonov et al., filed Jan. 18, 2000,(PCT/US00/01202); “METHODS OF POPULATING DATA STRUCTURES FOR USE INEVOLUTIONARY SIMULATIONS” by Selifonov and Stemmer (PCT/US00/01138),filed Jan. 18, 2000; and, e.g., “METHODS FOR MAKING CHARACTER STRINGS,POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” bySelifonov et al., filed Jul. 18, 2000 (U.S. Ser. No. 09/618,579). Theseprocedures are especially amenable to use in the automated systems andmethods herein.

For example, in silico methods of recombination can be effected in whichgenetic algorithms (GAs) or genetic operators (GOs) are used in acomputer to recombine sequence strings which correspond to homologous(or even non-homologous) nucleic acids. The resulting recombinedsequence strings are optionally converted into nucleic acids bysynthesis of nucleic acids which correspond to the recombined sequences,e.g., in concert with oligonucleotide synthesis/gene reassemblytechniques. This approach can generate random, partially random ordesigned variants. Many details regarding in silico recombination,including the use of genetic algorithms, genetic operators and the likein computer systems, combined with generation of corresponding nucleicacids (and/or proteins), as well as combinations of designed nucleicacids and/or proteins (e.g., based on cross-over site selection) as wellas designed, pseudo-random or random recombination methods are describedin “METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDESHAVING DESIRED CHARACTERISTICS” by Selifonov et al., filed Jan. 18,2000, (PCT/US00/01202) “METHODS OF POPULATING DATA STRUCTURES FOR USE INEVOLUTIONARY SIMULATIONS” by Selifonov and Stemmer (PCT/US00/01138),filed Jan. 18, 2000; and, e.g., “METHODS FOR MAKING CHARACTER STRINGS,POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” bySelifonov et al., filed Jul. 18, 2000 (U.S. Ser. No. 09/618,579).Extensive details regarding in silico recombination methods are found inthese applications.

Many methods of accessing natural diversity, e.g., by hybridization ofdiverse nucleic acids or nucleic acid fragments to single-strandedtemplates, followed by polymerization and/or ligation to regeneratefull-length sequences, optionally followed by degradation of thetemplates and recovery of the resulting modified nucleic acids can besimilarly used. In one method employing a single-stranded template, thefragment population derived from the genomic library(ies) is annealedwith partial, or, often approximately full length, ssDNA or RNAcorresponding to the opposite strand. Assembly of complex chimeric genesfrom this population is then mediated by nuclease-base removal ofnon-hybridizing fragment ends, polymerization to fill gaps between suchfragments and subsequent single stranded ligation. The parentalpolynucleotide strand can be removed by digestion (e.g., if RNA oruracil-containing), magnetic separation under denaturing conditions (iflabeled in a manner conducive to such separation) and other availableseparation/purification methods. Alternatively, the parental strand isoptionally co-purified with the chimeric strands and removed duringsubsequent screening and processing steps. Additional details regardingthis approach are found, e.g., in “SINGLE-STRANDED NUCLEIC ACIDTEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION” byAffholter, U.S. Ser. No. 09/656,549, filed Sep. 6, 2000. Further detailson adaptation of these methods to the present invention are found supra.

In another approach, single-stranded molecules are converted todouble-stranded DNA (dsDNA) and the dsDNA molecules are bound to a solidsupport by ligand-mediated binding. After separation of unbound DNA, theselected DNA molecules are released from the support and introduced intoa suitable host cell to generate a library enriched sequences whichhybridize to the probe. A library produced in this manner provides adesirable substrate for further diversification using any of theprocedures described herein. Further details on this approach areprovided herein.

Any of the preceding general mutation or recombination formats can bepracticed in a reiterative fashion (e.g., one or more cycles ofmutation/recombination or other diversity generation methods, optionallyfollowed by one or more selection methods) to generate a more diverseset of recombinant nucleic acids.

In general, the above references provide many basic mutation andrecombination formats as well as many modifications of these formats.Regardless of the format which is used, the nucleic acids of theinvention can be recombined (with each other or with related (or evenunrelated) to produce a diverse set of recombinant nucleic acids,including, e.g., sets of homologous nucleic acids.

Following recombination and/or other forms of mutation, any nucleicacids which are produced can be selected for a desired activity. In thecontext of the present invention, this can include testing for andidentifying any activity that can be detected in an automatable format,by any of the assays in the art. A variety of related (or evenunrelated) properties can be assayed for, using any available assay.These methods are automated according to the present invention asdescribed herein. As noted, DNA recombination and other forms ofmutagenesis, separately or in combination, provide robust, widelyapplicable, means of generating diversity useful for the engineering ofnucleic acids, proteins, pathways, cells and organisms to provide new orimproved characteristics.

It is often desirable to combine multiple diversity generatingmethodologies when generating diversity. For example, in conjunctionwith (or separately from) shuffling methods, a variety of mutationmethods can be practiced and the results (i.e., diverse populations ofnucleic acids) screened for in the systems of the invention. Additionaldiversity can be introduced by methods which result in the alteration ofindividual nucleotides or groups of contiguous or non-contiguousnucleotides, i.e., mutagenesis methods. Further details on certainexample mutation methodologies are provided below.

In one aspect, error-prone PCR is used, in which, e.g., PCR is performedunder conditions where the copying fidelity of the DNA polymerase islow, such that a high rate of point mutations is obtained along theentire length of the PCR product. Examples of such techniques are foundin the references above and, e.g., in Leung et al., (1989) Technique,1:11-15 (1989) and Caldwell et al. (1992) PCR Methods Applic. 2:28-33.Similarly, assembly PCR can be used, in a process which involves theassembly of a PCR product from a mixture of small DNA fragments. A largenumber of different PCR reactions can occur in parallel in the samevial, with the products of one reaction priming the products of anotherreaction. Sexual PCR mutagenesis can be used in which homologousrecombination occurs between DNA molecules of different but related DNAsequence in vitro, by random fragmentation of the DNA molecule based onsequence homology, followed by fixation of the crossover by primerextension in a PCR reaction. This process is described in the referencesabove, e.g., in Stemmer (1994) PNAS 91:10747-10751. Recursive ensemblemutagenesis can be used in which an algorithm for protein mutagenesis isused to produce diverse populations of phenotypically related mutantswhose members differ in amino acid sequence. This method uses a feedbackmechanism to control successive rounds of combinatorial cassettemutagenesis. Examples of this approach are found in Arkin and YouvanPNAS USA 89:7811-7815 (1992).

As noted, oligonucleotide directed mutagenesis can be used in a processwhich allows for the generation of site-specific mutations in any clonedDNA segment of interest. Examples of such techniques are found in thereferences above and, e.g., in Reidhaar-Olson et al. (1988) Science,241:53-57. Similarly, cassette mutagenesis can be used in a processwhich replaces a small region of a double stranded DNA molecule with asynthetic oligonucleotide cassette that differs from the nativesequence. The oligonucleotide can contain, e.g., completely and/orpartially randomized native sequence(s).

In vivo mutagenesis can be used in a process of generating randommutations in any cloned DNA of interest which involves the propagationof the DNA, e.g., in a strain of E. coli that carries mutations in oneor more of the DNA repair pathways. These “mutator” strains have ahigher random mutation rate than that of a wild-type parent. Propagatingthe DNA in one of these strains will eventually generate randommutations within the DNA.

Exponential ensemble mutagenesis can be used for generatingcombinatorial libraries with a high percentage of unique and functionalmutants, where small groups of residues are randomized in parallel toidentify, at each altered position, amino acids which lead to functionalproteins. Examples of such procedures are found in Delegrave and Youvan(1993) Biotechnology Research, 11:1548-1552. Similarly, random andsite-directed mutagenesis can be used. Examples of such procedures arefound in Arnold (1993) Current Opinion in Biotechnology, 4:450-455.

Many kits for mutagenesis are also commercially available. For example,kits are available from, e.g., Stratagene (e.g., the QuickChangesite-directed mutagenesis kit; and the Chameleon double-stranded,site-directed mutagenesis kit), Bio/Can Scientific, Bio-Rad (e.g., usingthe Kunkel method described above), Boehringer Mannheim Corp., ClonetechLaboratories, DNA Technologies, Epicentre Technologies (e.g., 5 prime 3prime kit); Genpak Inc, Lemargo Inc, Life Technologies (Gibco BRL), NewEngland Biolabs, Pharmacia Biotech, Promega Corp., QuantumBiotechnologies, Amersham International plc (e.g., using the Ecksteinmethod above), and Anglian Biotechnology ltd (e.g., using theCarter/Winter method above).

Any of the described shuffling or mutagenesis techniques can be used inconjunction with procedures which introduce additional diversity into agenome, e.g. a eukaryotic or bacterial genome. For example, in additionto the methods above, techniques have been proposed which producechimeric nucleic acid multimers suitable for transformation into avariety of species, including E. coli and B. subtilis (see, e.g.,Schellenberger U.S. Pat. No. 5,756,316 and the references above). Whensuch chimeric multimers consist of genes that are divergent with respectto one another, (e.g., derived from natural diversity or throughapplication of site directed mutagenesis, error prone PCR, passagethrough mutagenic bacterial strains, and the like), are transformed intoa suitable host, this provides a source of nucleic acid diversity forDNA diversification.

In one aspect, a multiplicity of monomeric polynucleotides sharingregions of partial sequence similarity can be transformed into a hostspecies and recombined in vivo by the host cell. Subsequent rounds ofcell division can be used to generate libraries, members of which,include a single, homogenous population, or pool of monomericpolynucleotides. Alternatively, the monomeric nucleic acid can berecovered by standard techniques, e.g., PCR and/or cloning, andrecombined in any of the recombination formats, including recursiverecombination formats, described above.

Methods for generating multispecies expression libraries have beendescribed (in addition to the reference noted above, see, e.g., Petersonet al. (1998) U.S. Pat. No. 5,783,431 “METHODS FOR GENERATING ANDSCREENING NOVEL METABOLIC PATHWAYS,” and Thompson, et al. (1998) U.S.Pat. No. 5,824,485 METHODS FOR GENERATING AND SCREENING NOVEL METABOLICPATHWAYS) and their use to identify protein activities of interest hasbeen proposed (In addition to the references noted above, see, Short(1999) U.S. Pat. No. 5,958,672 “PROTEIN ACTIVITY SCREENING OF CLONESHAVING DNA FROM UNCULTIVATED MICROORGANISMS”). Multispecies expressionlibraries include, in general, libraries comprising cDNA or genomicsequences from a plurality of species or strains, operably linked toappropriate regulatory sequences, in an expression cassette. The cDNAand/or genomic sequences are optionally randomly ligated to furtherenhance diversity. The vector can be a shuttle vector suitable fortransformation and expression in more than one species of host organism,e.g., bacterial species, eukaryotic cells. In some cases, the library isbiased by preselecting sequences which encode a protein of interest, orwhich hybridize to a nucleic acid of interest. Any such libraries can beprovided as substrates for any of the methods herein described.

Chimeric multimers transformed into host species are suitable assubstrates for in vivo shuffling protocols. Alternatively, amultiplicity of polynucleotides sharing regions of partial sequencesimilarity can be transformed into a host species and recombined in vivoby the host cell. Subsequent rounds of cell division can be used togenerate libraries, members of which, comprise a single, homogenouspopulation of monomeric or pooled nucleic acid. Alternatively, themonomeric nucleic acid can be recovered by standard techniques andrecursively recombined in any of the described shuffling formats.

Chain termination methods of diversity generation have also beenproposed (see, e.g., U.S. Pat. No. 5,965,408 and the references above).In this approach, double stranded DNAs corresponding to one or moregenes sharing regions of sequence similarity are combined and denatured,in the presence or absence of primers specific for the gene. The singlestranded polynucleotides are then annealed and incubated in the presenceof a polymerase and a chain terminating reagent (e.g., uv, gamma orX-ray irradiation; ethidium bromide or other intercalators; DNA bindingproteins, such as single strand binding proteins, transcriptionactivating factors, or histones; polycyclic aromatic hydrocarbons;trivalent chromium or a trivalent chromium salt; or abbreviatedpolymerization mediated by rapid thermocycling; and the like), resultingin the production of partial duplex molecules. The partial duplexmolecules, e.g., containing partially extended chains, are thendenatured and reannealed in subsequent rounds of replication or partialreplication resulting in polynucleotides which share varying degrees ofsequence similarity and which are chimeric with respect to the startingpopulation of DNA molecules. Optionally, the products or partial poolsof the products can be amplified at one or more stages in the process.Polynucleotides produced by a chain termination method, such asdescribed above are suitable substrates for DNA shuffling according toany of the described formats.

Diversity can also be generated using, for example, incrementaltruncation for the creation of hybrid enzymes (ITCHY) described inOstermeier et al. (1999) “A combinatorial approach to hybrid enzymesindependent of DNA homology” Nature Biotech 17:1205, can be used togenerate an initial recombinant library which serves as a substrate forone or more rounds of in vitro or in vivo shuffling methods. Anyhomology or non-homology based mutation/recombination format can be usedto generate diversity, separately or in combination.

In some applications, it is desirable to preselect or prescreenlibraries (e.g., an amplified library, a genomic library, a cDNAlibrary, a normalized library, etc.) or other substrate nucleic acidsprior to shuffling, or to otherwise bias the substrates towards nucleicacids that encode functional products (shuffling procedures can also,independently have these effects). For example, in the case of antibodyengineering, it is possible to bias the shuffling process towardantibodies with functional antigen binding sites by taking advantage ofin vivo recombination events prior to DNA shuffling by any describedmethod. For example, recombined CDRs derived from B cell cDNA librariescan be amplified and assembled into framework regions (e.g., Jirholt etal. (1998) “Exploiting sequence space: shuffling in vivo formedcomplementarity determining regions into a master framework” Gene 215:471) prior to DNA shuffling according to, any of the methods describedherein.

Libraries can be biased towards nucleic acids which encode proteins withdesirable enzyme activities. For example, after identifying a clone froma library which exhibits a specified activity, the clone can bemutagenized using any known method for introducing DNA alterations,including, but not restricted to, DNA shuffling. A library comprisingthe mutagenized homologues is then screened for a desired activity,which can be the same as or different from the initially specifiedactivity. An example of such a procedure is proposed in U.S. Pat. No.5,939,250. Desired activities can be identified by any method known inthe art. For example, WO 99/10539 proposes that gene libraries can bescreened by combining extracts from the gene library with componentsobtained from metabolically rich cells and identifying combinationswhich exhibit the desired activity. It has also been proposed (e.g., WO98/58085) that clones with desired activities can be identified byinserting bioactive substrates into samples of the library, anddetecting bioactive fluorescence corresponding to the product of adesired activity using a fluorescent analyzer, e.g., a flow cytometrydevice, a CCD, a fluorometer, or a spectrophotometer.

Libraries can also be biased towards nucleic acids which have specifiedcharacteristics, e.g., hybridization to a selected nucleic acid probe.For example, application WO 99/10539 proposes that polynucleotidesencoding a desired activity (e.g., an enzymatic activity, for example: alipase, an esterase, a protease, a glycosidase, a glycosyl transferase,a phosphatase, a kinase, an oxygenase, a peroxidase, a hydrolase, ahydratase, a nitrilase, a transaminase, an amidase or an acylase) can beidentified from among genomic DNA sequences in the following manner.Single stranded DNA molecules from a population of genomic DNA arehybridized to a ligand-conjugated probe. The genomic DNA can be derivedfrom either a cultivated or uncultivated microorganism, or from anenvironmental sample. Alternatively, the genomic DNA can be derived froma multicellular organism, or a tissue derived therefrom. Second strandsynthesis can be conducted directly from a hybridization probe used inthe capture, with or without prior release from the capture medium or bya wide variety of other strategies known in the art. Alternatively, theisolated single-stranded genomic DNA population can be fragmentedwithout further cloning and used directly in a shuffling-based genereassembly process. In one such method the fragment population derivedthe genomic library(ies) is annealed with partial, or, oftenapproximately full length ssDNA or RNA corresponding to the oppositestrand. Assembly of complex chimeric genes from this population is themediated by nuclease-based removal of non-hybridizing fragment ends,polymerization to fill gaps between such fragments and subsequent singlestranded ligation. The parental strand can be removed by digestion (ifRNA or uracil-containing), magnetic separation under denaturingconditions (if labeled in a manner conducive to such separation) andother available separation/purification methods. Alternatively, theparental strand is optionally co-purified with the chimeric strands andremoved during subsequent screening and processing steps. As setdetailed, e.g., in “SINGLE-STRANDED NUCLEIC ACID TEMPLATE-MEDIATEDRECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION” by Affliolter, U.S.Ser. No. 60/186,482 filed Mar. 2, 2000, and U.S. Ser. No. 09/656,549,Filed Sep. 6, 2000 shuffling using single-stranded templates and nucleicacids of interest which bind to a portion of the template can also beperformed.

“Non-Stochastic” methods of generating nucleic acids and polypeptidesare proposed in Short “Non-Stochastic Generation of Genetic Vaccines andEnzymes” WO 00/46344. These methods, including proposed non-stochasticpolynucleotide reassembly and site-saturation mutagenesis methods can beapplied to the present invention as well. Random or semi-randommutagenesis using doped or degenerate oligonucleotides is also describedin, e.g., Arkin and Youvan (1992) “Optimizing nucleotide mixtures toencode specific subsets of amino acids for semi-random mutagenesis”Biotechnology 10:297-300; Reidhaar-Olson et al. (1991) “Randommutagenesis of protein sequences using oligonucleotide cassettes”Methods Enzymol. 208:564-86; Lim and Sauer (1991) “The role of internalpacking interactions in determining the structure and stability of aprotein” J. Mol. Biol. 219:359-76; Breyer and Sauer (1989) “Mutationalanalysis of the fine specificity of binding of monoclonal antibody 51Fto lambda repressor” J. Biol. Chem. 264:13355-60); and “Walk-ThroughMutagenesis” (Crea, R; U.S. Pat. Nos. 5,830,650 and 5,798,208, and EPPatent 0527809 B1.

In one approach, described in more detail herein, single-strandedmolecules are converted to double-stranded DNA (dsDNA) and the dsDNAmolecules are bound to a solid support by ligand-mediated binding. Afterseparation of unbound DNA, the selected DNA molecules are released fromthe support and introduced into a suitable host cell to generate alibrary enriched sequences which hybridize to the probe. A libraryproduced in this manner provides a desirable substrate for any of theshuffling reactions described herein.

It will further be appreciated that any of the above describedtechniques suitable for enriching a library prior to shuffling can beused to screen the products generated by the methods of DNA shuffling.

The above references provide many mutational formats, includingrecombination, recursive recombination, mutation by non-recombinationdirected methods, recursive mutation in any format as well as manymodifications of these formats. Regardless of the diversity generationformat that is used, the nucleic acids of the invention can berecombined (with each other, or with related (or even unrelated)sequences) to produce a diverse set of recombinant nucleic acids,including, e.g., sets of homologous nucleic acids, as well ascorresponding polypeptides.

Non-PCR Based Recombination Methods

As noted above, site-directed or oligonucleotide-directed mutagenesismethods can be used to generate chimeras between 2 or more parentalgenes. Many methods are described in the literature and some are listedherein that do not depend on PCR, though PCR-based methods are alsofully described herein and useful in the context of the presentinvention.

A common theme to many non-PCR based methods is preparation of asingle-stranded template to which primers (e.g., syntheticoligonucleotides, single-stranded DNA or RNA fragments) are annealed,then elongated by a DNA or RNA polymerase in the presence of dNTPs andappropriate buffer. The gapped duplex can be sealed with DNA ligaseprior to transformation or electroporation into E. coli. In someinstances, e.g., where a substantially coextensive heterolog isgenerated by annealing of multiple primers to a template, ligase aloneis sufficient to produce a recombinant DNA strand. In some instances,e.g., where there are “flaps” of nucleic acid which do not hybridize tothe template, an exo- or endo-nuclease can be used to eliminateunhybridized portions of a bound nucleic acid prior to polymerase and/orligase treatment.

The newly synthesized strand is replicated and generates a chimeric genewith contributions from the oligo in the context of the single-stranded(ss) parent. The ss template can be prepared, e.g., by incorporation ofthe phage IG region into the plasmid and use of a helper phage such asM13KO7 or R408 to package ss plasmids into filamentous phage particles.The ss template can also be generated by denaturation of adouble-stranded template and annealing in the presence of the primers.Methods vary, e.g., in the enrichment protocols for isolation of thenewly synthesized chimeric strand over the parental template strand andare described in the references below. The “Kunkel” method usesuracil-containing templates. The Eckstein method usesphosphorothioate-modified DNA. The use of restriction selection orpurification can be used in conjunction with mismatch repair deficientstrains.

In the context of the present invention, the “mutagenic” primerdescribed in these methods can be one or more synthetic oligonucleotidesencoding any type of randomization, insertion, deletion, family geneshuffling oligonucleotides based on sequence diversity of homologousgenes, etc. Oligos that randomize particular sequences (e.g. NNG/C),encode conservative replacements for particular residues (e.g. NUN forhydrophobic residues), spiked oligos where the correct nucleotidesequence is synthesized in the background of a low level of all 3mismatched nucleotides, incorporation of deoxyinosine or other ambiguousnucleotide analogs, incorporation, insertions, deletions, error pronePCR, etc. can be used. The primer(s) can also be, e.g., fragments ofhomologous genes that are annealed to the ss parent template. In thisway chimeras between 2 or more parental genes can be generated.

Multiple primers can anneal to a given template and be extended tocreate multiply chimeric genes. The use of a DNA polymerase such asthose from phages T4 or T7 are good for this purpose as they will notdegrade or displace a downstream primer from the template.

In one class of preferred embodiments, the ss template or one or moreprimers (e.g., mutagenic primers) is immobilized on a solid substratesuch as a chip or a membrane. In other embodiments, annealing andextension occurs in a liquid phase array, such as in a reaction solutionwithin wells of a microtiter plate or an arrangement of test tubes.

Example: Dna Shuffling Using Uracil Containing Templates

For example, in one aspect, a gene of interest is cloned into an E. coliplasmid containing the filamentous phage intergenic (IG, ori) region.Single stranded (ss) plasmid DNA is packaged into phage particles uponinfection with a helper phage such as M13KO7 (Pharmacia) or R408 and ispurified by methods such as phenol/chloroform extraction and ethanolprecipitation. If this DNA is prepared in a dut⁻ ung⁻ strain of E. coli,a small number of uracil residues are incorporated into it in place ofnormal thymine residues. The ratio of the amount of uracil residues tothe amount of thymidine residues used typically depends on the desirednucleic acid fragment size. The ratio is optionally calculated usingappropriate software or instruction sets as described below. Theinstructions are typically programmed into a diversity generation deviceof the invention, e.g., in a computer readable format in a computeroperably coupled to a diversity generation device or directly into athermocycler used in a diversity generation device.

One or more primers as defined above are annealed to the ssuracil-containing template by heating to 90° C. and slowly cooling toroom temperature. An appropriate buffer containing all 4deoxyribonucleotides, T7 DNA polymerase and T4 DNA ligase is added tothe annealed template/primer mix and incubated between room temperature−37° C. for ≧1 hour. The T7 DNA polymerase extends from the 3′ end ofthe primer and synthesizes a complementary strand to the templateincorporating the primer. DNA ligase seals the gap between the 3′ end ofthe newly synthesized strand and the 5′ end of the primer. If multipleprimers are used, then the polymerase will extend to the next primer,stop (preferentially, polymerases that are arrested by downstream boundnucleic acids are used for this purpose) and ligase will seal the gap.As noted above, an exonuclease can be employed, e.g., prior topolymerase treatment.

The products of these reactions are then transformed into an ung⁺ strainof E. coli and antibiotic selection for the plasmid is applied. UracilN-glycosylase (the ung gene product) enzyme in the host cell recognizesthe uracil in the template strand and removes it, creating apyrimidinicsites that are either not replicated or which are corrected by the hostrepair systems using the newly synthesized strand as a template. Theresulting plasmids predominantly contain the desired change in the geneif interest. If multiple primers are used then it is possiblesimultaneously to introduce numerous changes in a single reaction. Ifthe primers are derived from fragments of homologous genes, thenmultiply chimeric genes can be generated.

Any of these diversity generating methods (shuffling, mutagenesis, etc.)can be combined with each other, in any combination selected by theuser, to produce nucleic acid diversity, which may be screened for usingany available screening method. The section below entitled “DiversityGeneration Modules” provides further details regarding generation ofdiversity in the devices, modules and systems of the present invention.

A. Diversity Generation Modules

The automated production of diverse libraries can be used to increasethe throughput of forced evolution methods. A variety of diversityproduction strategies can be used. Shuffling and other diversitygenerating modules of the invention provide a convenient way to generatediversity from starting nucleic acids. Diversity generation modulesautomate one or more relevant diversity generating process.

For example, the diversity generation module can take the form of anucleic acid shuffling or mutagenesis module which can accept inputnucleic acids or character strings corresponding to input nucleic acidsand can manipulate the input nucleic acids or the character stringscorresponding to input nucleic acids to produce output nucleic acids. Inaddition, the diversity generation modules of the invention areoptionally used to select appropriate input nucleic acids or characterstrings corresponding to input nucleic acids which are typicallyshuffled to produce output nucleic acids. In any case, the outputnucleic acids can comprise the one or more shuffled or mutagenizednucleic acids in the reaction mixture arrays of the invention, orfragments thereof. In addition to performing diversity-generationreactions, the diversity generation module optionally separates,identifies, purifies, immobilizes or otherwise treats diversifiednucleic acids for further analysis.

Common formats for the diversity generation module can include computersystems for designing and selecting nucleic acids, oligonucleotidesynthesizers, liquid handlers for moving and mixing reagents (e.g.,microwell plates, automatic pipettors, peristaltic pumps, etc.). Thenucleic acid shuffling module can include one or more microscale channelthrough which a shuffling reagent or product is flowed which can beintegrated in a chip, or present in a series of microcapillaries.

For example, in addition to, in conjunction with, or in place of astandard automatic pipetting station and set of microwell plates,devices or integrated systems can include physical or logical arrays ofreaction mixtures incorporated into the automatic pipetting station andset of microwell plates, or into a microscale device. Alternately, atleast one of the reaction mixtures can be incorporated into a microscaledevice or a delivery system which interfaces with the automaticpipetting station and set of microwell plates. In one embodiment, theone or more shuffled or mutagenized nucleic acids (or a transcribed formthereof) can be found within a microscale device or the microwellplates, or the one or more in vitro transcription or translationreagents can be found within the plates or the microscale device. Anyreagent associated with any operation of the module can be found withinstandard robotic systems, or in a microscale device, or in microwellplates, or on solid substrates or other storage systems as noted hereinand any operation or set of operations for the module can be performedin a microscale or milliscale format. Thus, all or part of the modulecan be embodied in one or more automatic pipetting station, roboticfluid handling systems, in microcapillary systems (e.g., includingintegrated microchannel devices). or combinations thereof.

(1.) Selection and Acquisition of Targets for Diversity GenerationProcesses

The identification and acquisition of nucleic acid targets for diversitygeneration can be performed by the diversity generating modules of theinvention. For example, selection algorithms can be used to identifysequences in public or proprietary databases which meet anyuser-selected criterion as a target for diversity generation. These usercriteria include activity, encoded activity, homology, publicavailability, and any other criteria of interest. In addition, characterstrings corresponding to nucleic acids (or their derived polypeptides)can be generated according to any set of criteria selected by the user,including similarity to existing sequences, modification of an existingsequence according to any desired modification parameter (geneticalgorithm, etc.), random or non-random (e.g., weighted) sequencegeneration, etc. Data structures comprising diverse sequences can beformed in a digital or analog computer or in a computer readable mediumand the data structures converted from character strings to nucleicacids (e.g., via automated synthesis protocols) for subsequent physicalmanipulations. Alternatively, the character strings are manipulated orshuffled “in silico” to produce diverse nucleic acids, based upon anygenetic algorithm or operator selected by the practitioner.

Either computer data or nucleic acids can be “data structures,” a termwhich refers to the organization and optionally associated device forthe storage of information, typically comprising multiple “pieces” ofinformation. The data structure can be a simple recordation of theinformation (e.g., a list) or the data structure can contain additionalinformation (e.g., annotations) regarding the information containedtherein, can establish relationships between the various “members”(information “pieces”) of the data structure, and can provide pointersor be linked to resources external to the data structure. The datastructure can be intangible but is rendered tangible whenstored/represented in tangible medium (e.g., in a computer medium, anucleic acid or set of nucleic acids, or the like). The data structurecan represent various information architectures including, but notlimited to simple lists, linked lists, indexed lists, data tables,indexes, hash indices, flat file databases, relational databases, localdatabases, distributed databases, thin client databases, and/or thelike.

Nucleic acids can be selected by the user based upon sequence similarityto one or more additional nucleic acid. Different types of similarityand considerations of various stringency and character string length canbe detected and recognized in the target acquisition phase of theinvention. For example, many homology determination methods have beendesigned for comparative analysis of sequences of biopolymers, forspell-checking in word processing, and for data retrieval from variousdatabases. With an understanding of double-helix pair-wise complementinteractions among the principal nucleobases in natural polynucleotides,models that simulate annealing of complementary homologouspolynucleotide strings can also be used as a foundation of sequencealignment or other operations typically performed on the characterstrings corresponding to the sequences of interest (e.g.,word-processing manipulations, construction of figures comprisingsequence or subsequence character strings, output tables, etc.). Anexample of a dedicated software package with genetic algorithms forcalculating sequence similarity and other operations of interest isBLAST, which can be used in the present invention to select targetsequence (e.g., based upon homology) for acquisition and supply to thediversity generating modules of the invention.

BLAST is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm firstidentifies high scoring sequence pairs (HSPs) by identifying short wordsof length W in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid (protein) sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

An additional example of a useful sequence alignment algorithm isPILEUP. PILEUP creates a multiple sequence alignment from a group ofrelated sequences using progressive, pairwise alignments. It can alsoplot a tree showing the clustering relationships used to create thealignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The methodused is similar to the method described by Higgins & Sharp, CABIOS5:151-153 (1989). The program can align, e.g., up to 300 sequences of amaximum length of 5,000 letters. The multiple alignment procedure beginswith the pairwise alignment of the two most similar sequences, producinga cluster of two aligned sequences. This cluster can then be aligned tothe next most related sequence or cluster of aligned sequences. Twoclusters of sequences can be aligned by a simple extension of thepairwise alignment of two individual sequences. The final alignment isachieved by a series of progressive, pairwise alignments. The programcan also be used to plot a dendogram or tree representation ofclustering relationships. The program is run by designating specificsequences and their amino acid or nucleotide coordinates for regions ofsequence comparison.

As noted, the diversity generation module can comprise a DNA shufflingmodule. In one preferred embodiment, this module accepts input nucleicacids such as DNAs or character strings corresponding to input DNAs andmanipulates the input DNAs or the character strings corresponding toinput DNAs to produce output DNAs, which output DNAs comprise the one ormore shuffled DNAs in the reaction mixture array. This can be performedby physical manipulation of nucleic acids as noted above, or characterstrings in computer systems, or both. For example, in addition to simplyselecting nucleic acids of interest, computer systems can be used toproduce character strings which correspond to nucleic acid targets fordiversity generation. A variety of genetic algorithms for modifyingcharacter strings which correspond to biopolymers are set forth indetail in, e.g., “METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES& POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” by Selifonov et al.,filed Feb. 5, 1999 (U.S. Ser. No. 60/118,854), U.S. Ser. No. 09/416,375filed Oct. 12, 1999, Application No. PCT/US00/01202, filed Jan. 18,2000, and, e.g., U.S. Ser. No. 09/618,579 filed Jul. 18, 2000. Thesegenetic algorithms (GAs) include, e.g., modifying nucleic acid sequencesto correspond to physical mutation events such as point mutation,nucleotide insertion, deletion, recombination and the like. Sequencescan also be tested for fitness or any other parameter, includingmultidimensional parameters, by parameterizing any selection criteriaand then selecting sequences which fall within the hyperspace defined bythe set of parameters. Combinations of automated design (e.g., proteindesign automation, or “PDA”), e.g., to select cross-over points forrecombination based upon, e.g., physical (e.g., presence of encodedprotein or other domains) or statistical (e.g., principal componentanalysis (“PCA”), Markov modeling, neural networks, etc.) criteria andrandom approaches (e.g., physical recombination of synthesized nucleicacids) can also be used. Further details on such approaches are found inthe applications noted above.

For example, the present methods for selecting nucleic acids forshuffling are used to insure that the parental sequences chosen fordiversity generation supply sufficient diversity yet can be recombinedor shuffled in practice. Typically, sequences are chosen forrecombination/shuffling based on percent homology, or based onphylogenetic relationships. Typically, a level of at least 50% sequencehomology is required for efficient recombination between a pair ofsequences. However, this general limit can be overcome by theintroduction of additional (wild type, naturally occurring or synthetic)sequences which the ‘bridge’ the diversity within any given sequencepair. This module may act to enhance recombinational efficiency within asequence population by further prescribing the synthesis or addition ofa limited set of additional sequences not resident within the initialparental sequences. The likelihood that any two or more parents arecompatible for recombination/shuffling is a consequence of the chance ofrecombination occurring during the process. Frequency of recombinationis a direct consequence of the melting point of the hybrid molecule.Phylogenetic relationship and/or percent homology provide indirectmeasurements of the same thing. Therefore, the following method isoptionally used to provide an improved selection of sequences fordiversity generation. The method is an automated process by whichparental sequences are found, scored and chosen for shuffling based onmelting temperature. In addition, parental divergence is calculated andscored to enable an experimenter to make an informed decision uponchoosing parental nucleic acids for shuffling.

In one embodiment, a set of nucleic acid sequences or character stringscorresponding to nucleic acid sequences is selected using a computer orset of instructions embodied in a computer readable medium, e.g., on aweb page. Such a method typically comprises performing an alignment,e.g., a pairwise alignment, between two or more potential parentalnucleic acid sequences, e.g., using clustalw or one or more of theprograms described herein. Potential parental nucleic acid sequences arealso optionally selected using a computer, e.g., by searching one ormore database for one or more nucleic acid sequence of interest and oneor more homolog of the one or more nucleic acid sequence of interest.

The number of mismatches between the alignment is then calculated.Melting temperatures for one or more window of w bases in the alignmentare also calculated, identifying those windows having a meltingtemperature greater than x. Melting temperatures are optionallycalculated from one or more set of empirical data or one or more meltingtemperature prediction algorithm. A window of w bases typicallycomprises, e.g., about 21 bases. Preferably, w is an odd number and themelting temperature cutoff, x, is typically about 65° C.

One or more crossover segment in the alignment is then identified. Acrossover segment is one comprising two or more windows having a meltingtemperature greater than x, which two or more windows are separated byno more than n nucleotides, with n typically about 2. FIG. 33illustrates the melting temperature for a pairwise hybridization. Inthis example, the line indicates the melting temperature cutoff pointand the arrows indicate various crossover segments.

The dispersion, e.g., the inverse of the average number of bases betweencrossover segments in the alignment, for the crossover segmentsidentified is then typically calculated. The above calculations are thencombined to provide two scores, e.g., a shuffleability score and adiversity capture score, for each alignment pair.

The shuffleability score is based on the number of windows having amelting temperature greater that x, the dispersion, and the number ofcrossover segments identified. For example, the number of windows, thedispersion, and the number of segments are multiplied together. Thisscore reflects how well the aligned sequences would cross over during ashuffling reaction, e.g., in silico shuffling or shuffling in anotherdiversity generation device of the invention, and how much of thesequences are likely to be shuffled.

The diversity capture score is based on the number of mismatches in thealignment, the number of windows having a melting temperature greaterthat x, the dispersion, and the number of crossover segments identified.The score is representative not only of how well the sequences wouldrecombine, but also of how well recombining these sequences togetherwould create diversity.

The sequences are then ranked according to one or both of the abovescores and sequences for shuffling are selected based on the ranks. Tofurther evaluate the sequences for shuffleability, the above steps areoptionally repeated, e.g., starting with the one or more parentalnucleic acid selected in the first cycle. Alternatively, the steps arerepeated starting with the same or different potential parental nucleicacid sequences using one or more different input parameters, e.g., forcalculating the melting temperature.

The above methods are optionally used, e.g., with varying potentialparental sequences and melting temperature parameters, e.g., to optimizethe diversity capture score while minimizing the amount of parentalsequences needed for shuffling. In addition, the algorithm is optionallyused with certain restrictions, e.g., that a particularly desirableparent or parents must be included in the final set of parents. Forexample, the method could be set up to walk between two parentalsequences of interest. “Walking” refers to the process by whichrecombinations are obtained between two low homology parental sequencesvia intermediate sequences, i.e., A recombines with B, which recombineswith C, which recombines with D, wherein A and D do not directlyrecombine.

Other parameters are also optionally optimized in the selection ofparents or to modify the scoring. Such parameters include, but are notlimited to, the activity of the various parents, freedom to operateclearance, e.g., by an automatic search through a patent or literaturedatabase, the feasibility of obtaining the parents, the expressionlevels of the parents, and the compatibility of the parents codingsequences with the codon bias of one or more organisms.

For example, the above method is optionally used as described below,e.g., in an automated computerized format. A researcher submits a smallmolecule substrate or product, e.g., to a computer program, e.g.,embodied in a diversity generation device or on a web page. A chemicalstructure comparison search is performed on the small molecule, e.g.,using ISIS or another such database. Such comparison is optionallyperformed manually or using a computer. The small molecule and relatedstructures or homologs are used to search one or more databases, e.g.,KEGG, WIT, or the like, for genes that are related to or have anactivity on one or more of the compounds of interest. The genes are usedto find homologs for shuffling, e.g., by searching databases, such asBLAST, HMMR, fasta, Smith Waterman, and the like. The gene sequencesfound are reverse translated, e.g., to optimize shuffleability, optimizecodon usage for a given host, and/or maximize the difference from aparent that is prohibited by a lack freedom to operate. In someembodiments, it is desirable to have as few genes as possible forshuffling. Therefore, the genes are optionally weighted based onactivity, species, environment, or diversity. A final set of parentalsequences is determined based on the scores obtained as described aboveand the various weights given to each sequence. Oligonucleotides orcharacter strings that correspond to oligonucleotides for gene synthesisbased on the selected parental nucleic acids are then created, e.g., forsynthetic shuffling or in silico shuffling.

Nucleic acids which hybridize to one another are often provided to thesystem as starting nucleic acids for recombination-based diversitygeneration procedures. Further, nucleic acid hybridization can beestimated and used as a basis for selection in a computer system, in amanner similar to selecting for sequence similarity as set forth above(similar sequences typically hybridize). Nucleic acids “hybridize” whenthey associate, typically in solution. Nucleic acids hybridize due to avariety of well characterized physico-chemical forces, such as hydrogenbonding, solvent exclusion, base stacking and the like and, thus, theseinteractions can be modeled. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part I chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays,” (Elsevier, New York), aswell as in Ausubel, supra. Hames and Higgins (1995) Gene Probes 1 IRLPress at Oxford University Press, Oxford, England, (Hames and Higgins 1)and Hames and Higgins (1995) Gene Probes 2 IRL Press at OxfordUniversity Press, Oxford, England (Hames and Higgins 2) provide detailson the synthesis, labeling, detection and quantification of DNA and RNA,including oligonucleotides.

“Stringent hybridization wash conditions” in the context of nucleic acidhybridization experiments such as Southern and northern hybridizationsare sequence dependent, and are different under different environmentalparameters. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993), supra. and in Hames and Higgins, 1 and 2. Forpurposes of the present invention, generally, “highly stringent”hybridization and wash conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of the test sequencehybridizes to a perfectly matched probe. Very stringent conditions areselected to be equal to the T_(m) for a particular probe.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of stringent wash conditions is a 0.2×SSC wash at65° C. for 15 minutes (see, Sambrook, supra for a description of SSCbuffer). Often the high stringency wash is preceded by a low stringencywash to remove background probe signal. An example low stringency washis 2×SSC at 40° C. for 15 minutes. In general, a signal to noise ratioof 5× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization. Comparative hybridization can be used to identify nucleicacids as inputs to the systems of the invention.

Providing nucleic acids which are identified or generated as noted aboveoptionally takes one of two basic forms.

First, where a nucleic acid is selected which corresponds to aphysically existant nucleic acid, that nucleic acid can be acquired bycloning, PCR amplification or other nucleic acid isolation methods as iscommon in the art. An introduction to such methods is found in availablestandard texts, including Berger and Kimmel, Guide to Molecular CloningTechniques Methods in Enzymology volume 152 Academic Press, Inc., SanDiego, Calif. (Berger); Sambrook et al., Molecular Cloning—A LaboratoryManual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989 (“Sambrook”) and Current Protocols in MolecularBiology, F. M. Ausubel et al., eds., Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(supplemented through 1999) (“Ausubel”)). Examples of techniquessufficient to direct persons of skill through in vitro amplificationmethods, useful in identifying, isolating and cloning nucleic aciddiversity targets, including the polymerase chain reaction (PCR) theligase chain reaction (LCR), Q∃-replicase amplification and other RNApolymerase mediated techniques (e.g., NASBA), are found in Berger,Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No.4,683,202; PCR Protocols A Guide to Methods and Applications (Innis etal. eds) Academic Press Inc. San Diego, Calif. (1990) (Innis); Amnheim &Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991)3, 81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173;Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell etal. (1989) J. Clin. Chem. 35, 1826; Landegren et al., (1988) Science241, 1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; Wu andWallace, (1989) Gene 4, 560; Barringer et al. (1990) Gene 89, 117, andSooknanan and Malek (1995) Biotechnology 13: 563-564. Improved methodsof cloning in vitro amplified nucleic acids are described in Wallace etal., U.S. Pat. No. 5,426,039. Improved methods of amplifying largenucleic acids by PCR are summarized in Cheng et al. (1994) Nature 369:684-685 and the references therein, in which PCR amplicons of up to 40kb are generated. One of skill will appreciate that essentially any RNAcan be converted into a double stranded DNA suitable for restrictiondigestion, PCR expansion and sequencing using reverse transcriptase anda polymerase. See, Ausubel, Sambrook and Berger, all supra.

Host cells can be transduced with nucleic acids of interest, e.g.,cloned into vectors, for production of nucleic acids and expression ofencoded molecules (these encoded molecules can be used, e.g., ascontrols to determine a baseline activity to compare encoded activitiesof a diverse library of nucleic acids to). In addition to Berger,Sambrook and Ausubel, a variety of references, including, e.g., Freshney(1994) Culture of Animal Cells a Manual of Basic Technique, thirdedition, Wiley-Liss, New York and the references cited therein, Payne etal. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds) (1995) Plant Cell,Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds)The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.provide additional details on cell culture, cloning and expression ofnucleic acids in cells.

Sources for physically existant nucleic acids include nucleic acidlibraries, cell and tissue repositories, the NIH, USDA and othergovernmental agencies, the ATCC, zoos, nature and many others familiarto one of skill. For example, a wide variety of samples can be obtainedfrom nature which are suitable for use in the present invention. Theseinclude, but are not limited to, environmental isolates from remote,unusual, contaminated or common soils, clays, aquifers and marinelocalities; high and low moisture environments; living, dead, decayed orpartially decayed tissues of plants or animals; environmental isolatescontaining a plurality of microorganisms; extracts from the gut flora ofvertebrates and invertebrates, including symbiotic and endosymbioticmicroorganisms. While these diverse sources provide many nucleic acids,there are many others which exist only as a result of computeralgorithms as described above, or, even though existant, are difficultto acquire from nature (but often straightforward to synthesize, givenan appropriate sequence).

The second basic method for acquiring nucleic acids does not rely on thephysical pre-existence of a nucleic acid. Instead, nucleic acids aregenerated synthetically, e.g., using well-established nucleic acidsynthesis methods. For example, nucleic acids can be synthesized usingcommercially available nucleic acid synthesis machines which utilizestandard solid-phase methods. Typically, fragments of up to about 100bases are individually synthesized, then joined (e.g., by enzymatic orchemical ligation methods, or polymerase mediated recombination methods)to form essentially any desired continuous sequence or sequencepopulation. For example, the polynucleotides and oligonucleotides of theinvention can be prepared by chemical synthesis using, e.g., theclassical phosphoramidite method described by Beaucage et al., (1981)Tetrahedron Letters 22:1859-69, or the method described by Matthes etal., (1984) EMBO J. 3: 801-05., e.g., as is typically practiced inautomated synthetic methods. According to the phosphoramidite method,oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer,assembled and, optionally, cloned in appropriate vectors. In addition,essentially any nucleic acid can be custom ordered from any of a varietyof commercial sources, such as The Midland Certified Reagent Company(mcrc®oligos.com), The Great American Gene Company, ExpressGen Inc.,Operon Technologies Inc. (Alameda, Calif.) and many others. Similarly,peptides and antibodies (useful in various embodiments noted below) canbe custom ordered from any of a variety of sources, such as PeptidoGenic(pkim@ccnet.com), HTI Bio-products, inc., BMA Biomedicals Ltd (U.K.),Bio.Synthesis, Inc., Research Genetics (Huntsville, Ala.) and manyothers.

Synthetic approaches to nucleic acid generation have the advantage ofeasy automation. Oligonucleotide synthesis machines can easily beinterfaced with a digital system that instructs which nucleic acids tobe synthesized (indeed, such digital interfaces are generally part ofstandard oligonucleotide synthesis devices). Similarly, ordering nucleicacids from commercial sources can be automated through simple computerprogramming and use of the internet (e.g., by having the user selectnucleic acids which are desired and providing an automated orderingsystem), with provisions for user inputs (nucleic acid selection) andoutputs (synthesis of nucleic acids which are ordered).

Synthetic approaches can also be used to automate simultaneous sequenceacquisition and diversity generation, i.e., through “oligonucleotideshuffling” and related technologies (see also, “OLIGONUCLEOTIDE MEDIATEDNUCLEIC ACID RECOMBINATION” by Crameri et al., filed Feb. 5, 1999 (U.S.Ser. No. 60/118,813) and filed Jun. 24, 1999 (U.S. Ser. No. 60/141,049)and filed Sep. 28, 1999 (U.S. Ser. No. 09/408,392, Attorney DocketNumber 02-29620US) and USSN PCT/US00/01203 filed Jan. 18, 2000; and “USEOF CODON-BASED OLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” byWelch et al., filed Sep. 28, 1999 (U.S. Ser. No. 09/408,393, AttorneyDocket Number 02-010070US); and “METHODS FOR MAKING CHARACTER STRINGS,POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” bySelifonov et al. filed Feb. 5, 1999 (U.S. Ser. No. 60/118,854), U.S.Ser. No. 09/416,375 filed Oct. 12, 1999, Application No. PCT/US00/01202,filed Jan. 18, 2000, and, e.g., U.S. Ser. No. 09/618,579 filed Jul. 18,2000). In these methods, nucleic acid oligonucleotides corresponding tomultiple parental nucleic acids are synthesized, mixed and assembled viapolymerase (e.g., PCR) or ligase (or both) mediated methods to producerecombinant nucleic acids which have subsequences corresponding tomultiple parental nucleic acid types.

(2.) Sources and Destinations for Nucleic Acids in the Module

The assays of the invention are optionally partially or completelyperformed in a flowing format. That is, nucleic acids or other relevantreaction reagents are optionally flowed from sources (wells, channels,oligonucleotide synthesis elements, etc.) to destinations (reactionwells, channels, arrays, etc.), with reactions optionally beingcontrolled by flowing reactants into contact in the system.

Thus, the nucleic acids which are selected and/or acquired optionallyinclude one or more sources of one or more nucleic acids whichcollectively or individually comprise a first population of nucleicacids. The diversified nucleic acids are produced by recombining orotherwise mutating one or more members of the first population ofnucleic acids. This source of nucleic acids can be an in vitro, in vivoor virtual (in a digital system, i.e., “in silico”) source.

Sources of nucleic acids can include at least one nucleic acid,including, e.g., any of: a synthetic nucleic acid, a DNA, an RNA, a DNAanalogue, an RNA analogue, a genomic DNA, a cDNA, an mRNA, an nRNA, anaptamer, a cloned nucleic acid, a cloned DNA, a cloned RNA, a plasmidDNA, a viral DNA, a viral RNA, a YAC DNA, a cosmid DNA, a BAC DNA, aP1-mid, a phage DNA, a single-stranded DNA, a double-stranded DNA, abranched DNA, a catalytic nucleic acid, an antisense nucleic acid, an invitro amplified nucleic acid, a PCR amplified nucleic acid, an LCRamplified nucleic acid, a Q∃-replicase amplified nucleic acid, anoligonucleotide, a nucleic acid fragment, a restriction fragment or anycombination thereof, or other nucleic acid forms which are available.Alternately, the sources can be virtual or virtual and synthetic, andcan include one or more character string corresponding to such sources.In addition to virtual sources, data structures (which can be physicalor virtual) can be sources of nucleic acids (e.g., by combiningcharacter strings with synthetic methods), including diversified nucleicacids.

In addition to a source of nucleic acid, the module can include apopulation destination region. During operation of the device, one ormore members of the first population are optionally moved from one ormore sources of the one or more nucleic acids to the one or moredestination regions.

In general, the devices and systems can include nucleic acid movementmeans for moving the one or more members from the one or more sources ofthe one or more nucleic acids to the one or more destination regions (avariety of fluidic and non-fluidic means of moving components aredescribed herein).

Sources, destinations and source and destination regions can bephysically embodied in many different ways. For example, they can bemicrotiter wells or dishes, fritted microtiter trays (e.g., for couplingto column chromatographic methods) microfluidic systems, microchannels,containers, data structures, computer systems, combinations thereof, orthe like. Examples of sources/destinations include solid phase arrays,liquid phase arrays, containers, microtiter trays, microtiter traywells, microfluidic components, microfluidic chips, test tubes,centrifugal rotors, microscope slides, an organism, a cell, a tissue,and combinations thereof.

As is noted in more detail herein, the systems of the invention also cansimilarly include sources of in vitro transcription or translationreagents, where, during operation of the device, the in vitrotranscription reagent or an in vitro translation reagent is flowed froma source into contact with nucleic acids to be transcribed/translated.Sources and destinations for other reactants as noted herein are alsooptionally provided.

Any of the operations to be performed on individual array members can beperformed sequentially or in parallel. As noted throughout, certainphysical array formats such as microtiter tray-based approaches are wellsuited to parallel operations (i.e., having the same or similaroperations performed by approximately simultaneous additions of relevantreagents to the array, or approximately simultaneous removal ofmaterials from the array (e.g., for re-plating (e.g., for arrayduplication), purification of materials, and/or other downstreamoperations. As discussed herein, conventional high-throughput roboticsprovide one convenient way of performing these operations, which may, ofcourse, also be provided by manual manipulations, microfluidicapproaches, or other available methods. In some array formats,sequential operations are more conveniently performed, e.g., where thearray is a logical array with members which are not located in formatsthat provide for parallel manipulations.

In either case, robotic or other manipulations can be performeduniformly to the array, or can be selectively performed to individualarray members. These manipulations, and the actual motions used toachieve selective or parallel manipulations can be controlled byappropriate controller devices, e.g., computers linked to roboticelements with software comprising instruction sets for regulating therobotic or other material manipulative elements. The software isoptionally user programmable, i.e., to provide for parallel or selectiveoperations, e.g., to select “hits” for further manipulations.

Generally, as noted herein, master arrays or data sets (or both) can bemaintained that preserve information regarding the spatial location ofarray elements in the system. Generally, duplicate arrays are acted uponby system elements (e.g., reagents are added to or material removed fromone or more duplicate array members), rather than the preserved masterarray members or data set elements.

In addition to flowable formats, nucleic acids, transcription reagents,translation reagents or other relevant reactants are optionally fixed atone or more sources or at one or more destination regions. In these“fixed” or “partially flowing” formats, reagents can be localized to oneor more locations and cognate reagents either fixed in proximity, orflowed (e.g., via pipetting) or otherwise delivered (e.g., viaaerosolization, lyophilization, etc.) into contact with reagents ofinterest.

Movement means for moving nucleic acids and other reagents include fluidpressure modulators (e.g., pipettors or other pressure-driven channelsystems), electrokinetic fluid force modulators, electroosmotic flowmodulators, electrophoretic flow modulators, centrifugal forcemodulators, robotic armatures, pipettors, conveyor mechanisms, steppermotors, robotic plate manipulators, peristaltic pumps, magnetic fieldgenerators, electric field generators, fluid flow paths and the like.

For example, the diversity generating module can include one or morerecombination modules which move one or more members of a population ofnucleic acids into contact with one another, thereby facilitatingrecombination of the first population of nucleic acids. Similarly, thediversity generation module can include one or more reaction mixturearraying modules, which move one or more of the one or more diverse(e.g., shuffled) nucleic acids into one or more spatial positions. Thesystem can also provide for moving in vitro transcription/translationreactant components into desired locations in the array of reactionmixtures.

(3.) Dilution/Concentration Module

Shuffling/recombination/diversification module(s), or other modulesherein, optionally include a dilution or concentration function. Inparticular, it is often desirable to normalize the level of reactant orproduct at an array position (e.g., in a duplicate diluted orconcentrated array) so that product activities can be directly comparedacross an array. This typically involves determining the concentrationof products (proteins, nucleic acids, etc.) or reactants (nucleic acids,transcription buffers, translation buffers, etc.) at sites in the arrayand diluting or concentrating the products or reactants appropriately.The dilution/concentration module or module function can form newdiluted arrays or can dilute reactants or products at array sites. Forexample, the dilution/concentration module can re-array amplifiedphysical or logical array of polypeptides or in vitro transcribednucleic acids in a secondary polypeptide or in vitro transcribed nucleicacid array which has an approximately uniform concentration ofpolypeptides or in vitro transcribed nucleic acids at a plurality oflocations in the secondary polypeptide array.

To be able easily to recover nucleic acids which encode products ofinterest, it is generally desirable to limit the number of differentnucleic acids at defined sites in an array. For example, when arrangedin a microtiter tray or other physical array, e.g., for subsequentamplification or processing it is useful to dilute or concentrate arraymembers to an average of approximately 0.1-100 nucleic acids (e.g.,unique nucleic acids) per well or other storage site. This isparticularly relevant at the start of the arraying process followinginitial extraction, mutagenesis or cloning of member nucleic acids.Typically, nucleic acids are arranged at about 1-10, and often at anaverage of approximately 1-10 or 1-5 nucleic acids per well prior toamplification. Subsequent amplification in preparation for arrayduplication can increase this by, e.g., about 2-about 100 fold or more.In contrast, subsequent amplification for purposes of conductingtranscription, translation and/or screening can increase theconcentration of member nucleic acids by, e.g., about >100-fold or more.

The diluter can operate prior to or after diversity generation orbetween any reaction steps. For example, one embodiment includes adiluter which pre-dilutes one or more shuffled or otherwise diversifiednucleic acids (e.g., by diluting members of a population with a bufferprior to arraying the members, e.g., in the reaction mixture arraysherein). In other aspects, the diluter dilutes nucleic acids as part ofproducing copy arrays from amplified arrays of nucleic acids.

Typical concentration ranges for diluted nucleic acids are in the rangeof about 0.01 to 100 molecules per microliter (although, in certainembodiments where lipid vesicles are used as reaction vessels, thisconcentration can be somewhat different, as described supra).

Typical dilution/concentration operations are performed by any availablemethod, including the addition of buffers (e.g., by pipetting),lyophilization, osmosis, precipitation, chromatography and the like.

In one example, DNA is diluted and aliquotted into wells such that theconcentration approaches a statistical approximation of the desiredconcentration. The DNA is fluorescently labeled, during or afterdiversity generation, followed by FACS or other fluorescence-based cellsorting. The sorting and isolation of individual DNA fragments isoptionally coupled to a dispensing device such as a fraction collectorsuch that a collection array (e.g., microtiter tray) receives about 1molecule/well. The DNA is affinity tagged such that, e.g., one affinitytag exists per molecule. Subsequent binding to an assay vehicle allows asingle dsDNA molecule to bind each compartment in the assay.

DNA tagging formats include, e.g., 5′ termini DNA/RNA labeling byaminotag phosphoramidites, such as those described in Olejnik et al.(1998) “Photocleavable Aminotag Phorphoramidites for 5′termini DNA/RNAlabeling” Nucleic Acids Res. 26(15):3572-3576, in which a photocleavableamine can be introduced on the 5′ terminal phosphate and conjugated witha variety of amine-reactive markers such as biotin, digoxigenin ortetramethylrhodamine. The assay vehicles for compartmentalization ofaffinity tagged dsDNA can bind the DNA to a derivatized microtiter platedirectly or to, e.g., beads which are subsequently dispensed at a rateof, e.g., one bead per well. The bound DNA can be used to isolatehybridizing fragments or other hybridizing shuffled variants.

More than one DNA fragment can be dispensed into separate wells, withthe diversity generation and assaying steps being run as small pools ofsamples of interest. In some cases, this partially pooled approach ispreferred, e.g., for assaying larger libraries of diversified nucleicacids, or where the cost of reagents (e.g., transcription/translationreagents) is limiting. However, there are some drawbacks to thisapproach, such as a dilution of average activity in the wells,inhibition of individual pool members by other members in the wells,etc.

(4.) Processing of Acquired Nucleic Acids to IncreaseDiversity—Fragmentation Based Methods

As noted, the nucleic acid diversity generation (e.g., shuffling) modulecan permit hybridization of the nucleic acid fragments followed byelongation with a polymerase which elongates the hybridized nucleicacid. Several (though not all) diversity generation methods relyinitially on the production of fragmented DNA. In general, one or moreshuffled nucleic acid(s) can be produced by synthesizing a set ofoverlapping oligonucleotides, or by cleaving a plurality of homologousnucleic acids to produce a set of cleaved homologous nucleic acids, orboth, and permitting recombination to occur between the set ofoverlapping oligonucleotides, the set of cleaved homologous nucleicacids, or a combined set of overlapping oligonucleotides and set ofcleaved homologous nucleic acids. Fragmented DNA is recombined, e.g.,taking advantage of hybridization and PCR or LCR gene reconstructionmethods described in the references above to produce full-length,diversified recombinant nucleic acid libraries. These libraries areoptionally screened for the expression of products of interest. Thus,the diversity module optionally fragments input nucleic acids to producenucleic acid fragments, or the input nucleic acids can themselvesinclude cleaved or synthetic nucleic acid fragments.

A number of automated approaches can be used to produce “fragmented”nucleic acids. Fragmented nucleic acids can be provided by mechanicallyshearing nucleic acids, by enzymatically or chemically cleaving nucleicacids, by partially synthesizing nucleic acids, by random primerextending or directed primer extending double-stranded orsingle-stranded nucleic acid templates, by incorporating cleavableelements into the nucleic acids during synthesis, or the like. Templatesor starting materials for such procedures include naturally occurringnucleic acids, synthetic nucleic acids, DNA in any form, RNA in anyform, DNA analogues, RNA analogues, genomic DNAs, cDNAs, mRNAs, nRNAs,cloned nucleic acids, cloned DNAs, cloned RNAs, plasmid DNAs, viralDNAs, viral RNAs, YAC DNAs, cosmid DNAs, branched DNAs, DNA and/or RNAisolated from heterogeneous microbial populations, catalytic nucleicacids, antisense nucleic acids, in vitro amplified nucleic acids, PCRamplified nucleic acids, LCR amplified nucleic acids, SDA nucleic acids,Q∃-replicase amplified nucleic acids, nucleic acid sequence-basedamplified (NASBA) nucleic acids, transcription-mediated amplified (TMA)nucleic acids, oligonucleotides, nucleic acid fragments, restrictionfragments, combinations thereof and any other available material.Nucleic acids can be partially or substantially purified prior tofragmentation, or can be unpurified.

For example, nucleic acids can be fragmented enzymatically, e.g., DNAcan be fragmented using a nuclease such as a DNAse. In the context ofthe present invention, a fragmentation module can include containerssuch as microtiter plates or microfluidic chips into which parentalnucleic acids (e.g., homologous DNAs) are dispensed, mixed andfragmented by the addition of DNAse. In addition, the fragmentationmodule is optionally operably coupled to a programmed thermocyclerand/or computer for directing fragmentation. For example, a computer isused to calculate conditions for fragmentation that produce desiredlength fragments. For example, when uracil incorporation and cleavage isused to produce nucleic acid fragments, a computer optionally calculatesthe amount of uracil residues to be used in relation to thymidineresidues, e.g., based on user input comprising the desired fragmentlength. The reaction is allowed to proceed for a selected period oftime, or in parallel reactions having different time periods, to produceone or multiple sets of nucleic acid fragments. The addition of DNAse orother cleavage enzymes can occur before or after dispensing the parentalnucleic acids into one or more systems which facilitate downstreamprocessing (e.g., prior to dispensing into microwell plates, microchips,or the like). The nucleic acid fragments can be contacted to one anotherin a single pool, or in multiple pools.

Alternately, or in combination, nucleic acids are mechanically sheared,e.g., by vortexing, sonicating, point-sink shearing or other similaroperations, before or after addition to the one or more systems whichfacilitate downstream processing. Mechanical shearing of nucleic acidshas the advantage of being largely sequence independent, which, attimes, is desirable, e.g. where no bias is desired in the shearednucleic acid fragments. For example, the point-sink shearing method isdescribed in Thorstenson et al., (1998) “An Automated HydrodynamicProcess for Controlled, Unbiased DNA shearing,” Genome Research8:848-855. Basically, this method consists of forcing a solution of DNAinto a narrowed region of a channel, putting sufficient force on the DNAto break it up. Although this method typically generated relativelylarge DNA fragments (500-1000 bp), the size of fragments can be reducedby increasing the velocity of the solution, decreasing the size of thechannel, vibrating the channel, e.g., at the channel entrance (e.g.,using a circular piezo-electric device), or the like.

In a second alternate embodiment, nucleic acids are “fragmented” bysynthesis of fragments (rather than cleavage) which correspond insequence to subsequences of one or more parental nucleic acids. Forexample, synthetic oligonucleotide “fragments” can be made in anautomatic synthesizer which correspond to any sequence of interest. Thismethod has the advantage of easy combination with in silico approaches(e.g., in silico recombination of character strings can be performed,followed by synthesis of the oligonucleotides which correspond to anydesired character string). Indeed, the oligonucleotides which aregenerated can provide any desired diversity in products which are formedusing the oligonucleotides—thus, sequence acquisition and at least afirst round of diversity generation can be performed simultaneously.Further details regarding Oligonucleotide synthetic approaches and “insilico” shuffling approaches are found in OLIGONUCLEOTIDE MEDIATEDNUCLEIC ACID RECOMBINATION” by Crameri et al., supra., and “USE OFCODON-BASED OLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” by Welchet al., supra., and “METHODS FOR MAKING CHARACTER STRINGS,POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” bySelifonov et al., supra., and further details on these methods are alsofound, supra.

In a third and also preferred embodiment, DNA fragmentation is achievedvia incorporation of cleavage targets into nucleic acids of interest. Inthis embodiment, modified nucleotides or other structures areincorporated into nucleic acids during synthesis (whether chemical,enzymatic, or both) of the nucleic acids. These modified nucleotides orother structures become cleavage points within a nucleic acid into whichthey are incorporated. One example of this approach is described, e.g.,in PCT US96/19256. As noted in the '256 application, nucleic acidsynthesis can be conducted to produce nucleic acids of interest (e.g.,via PCR, e.g., using a computer or computer program to calculate theuracil/thymidine ratio necessary to produce nucleic acid fragments of adesired size or synthetic methods), incorporating uracil into thenucleotides in a stochastic or directed fashion. The PCR products arethen fragmented by digestion with UDG-glycosylase, which forms strandbreaks at the uracil residues. Further details on this procedure arefound below.

Similarly, RNA nucleotides can be incorporated into DNA chains(synthetically or via enzymatic incorporation); these nucleotides thenserve as targets for cleavage via RNA endonucleases. A variety of othercleavable residues are known, including certain residues which arespecific or non-specific targets for enzymes, or other residues whichserve as cleavage points in response to light, heat or the like. Wherepolymerases are currently not available with activity permittingincorporation of a desired cleavage target, such polymerases can beproduced using shuffling methods to modify the activity of existingpolymerases, or to acquire new polymerase activities.

Simple chain termination methods can also be used to produce nucleicacid fragments, e.g., by incorporating dideoxy nucleotides into thereaction mixture(s) of interest.

In any case, once fragmentation is performed to the extent desired, thereaction is transferred to a recombination/resynthesis module. Thismodule optionally dispenses resulting elongated nucleic acids into oneor more multiwell plates, or onto one or more solid substrates, or intoone or more microscale systems, or into one or more containers forfurther operations by the system.

In one embodiment, diversity generation module(s) (or any other moduleherein) can include a fragment length purification portion whichpurifies selected length fragments of the nucleic acid fragments.Fragment purification can be performed by electrophoresis (e.g., gelelectrophoresis), column chromatography, incorporation of a label,incorporation of a purification tag, or any other currently availablemethod.

As noted above, the diversity module also optionally dilutes orconcentrates nucleic acids (e.g., produced by elongation of fragmentpopulations) and dispenses them. For example, elongated nucleic acidsproduced after PCR or ligase-mediated gene reconstruction can bedispensed into one or more multiwell plates or other arrayconfigurations at a selected density per well (or chamber, channel,container, etc., depending on the configuration) of the elongatednucleic acids. This dilution/concentration function is useful innormalizing assay results. That is, having array members at similar (orotherwise defined) concentrations permits analysis of results (e.g.,concentration or activity levels of products). Similarly, where productconcentrations are different, it is useful to dilute or concentrateproducts to similar or at least defined concentrations to facilitateresult interpretation.

In one embodiment, the device or integrated system includes a nucleicacid fragmentation module and a recombination region. The fragmentationmodule includes, e.g., a nuclease, a mechanical shearing device, apolymerase, a random primer, a directed primer, a nucleic acid cleavagereagent, a chemical nucleic acid chain terminator, an oligonucleotidesynthesizer, or other element for producing fragmented nucleic acids asdescribed above. During operation of the device, fragmented DNAs orother nucleic acids produced in the fragmentation module, are recombinedin the recombination region (a well, channel, chamber or other containeror substrate or surface) to produce one or more shuffled nucleic acids.

As noted, fragments (or full-length nucleic acids in other modulesherein) are often purified prior for further operations by the system.This purification incorporate any of the purification methods common toDNA or RNA purification, including electrophoresis (in gels, capillarychannels, etc.), chromatography or the like.

An Improved StEP

The effectiveness of DNA shuffling by staggered extension process (StEP)depends in certain formats in part on the rapidity of thermocyclingbetween denaturation and extension steps. Very rapid thermocycling canbe used to limit extension. The more limited the extension, the smallerthe resulting fragments and the finer the “granularity” of the resultingrecombination. Controlled incorporation of uracil into parentaltemplates with uracil glycosylase to generate AP sites are used toprovide an alternate method of controlling fragment size. Thegranularity of recombination is controlled, e.g., by the frequency ofapurinic sites in parental templates, as these sites serve asreplication terminators in the StEP reaction. A further improvement usesa thermostable uracil glycosylase and dUTP in the StEP reaction to addreplication terminators to newly synthesized DNA fragments, assuringrecombination throughout the StEP reaction.

Fragmentation Example: Ung-End Fragmentation: Use in Single-Tube DNAShuffling Reactions

This example describes single-tube DNA shuffling according to thepresent invention including simplification of DNase enzymaticfragmentation, size fractionation and purification of DNA by agarose gelelectrophoresis or other procedures. An alternative to laborious andhard-to-control standard fragmentation protocols includes the use ofcontrolled uracil incorporation into starting DNA, e.g., via PCR withdUTP, followed by fragmentation of the uracil-containing DNA with twoenzymes: Uracil N-Glycosylase (Ung) which hydrolyzes the n-glycosidicbond between the deoxyribose sugar and uracil to generate apurinic (orAP) sites, followed by the use of a 5′ AP endonuclease, such asEndonuclease IV (End) which cleaves a single strand of DNA 5′ to APsites, leaving a 3′-hydroxy-nucleotide and 5′-deoxyribose phosphatetermini. See also, Freidberg et al. (1995) DNA Repair and Mutagenesis.pp. 1-698. ASM Press. Washington, D.C.

A fundamental advantage of Ung-End fragmentation over DNAse I treatment,is that fragmentation is simply a function of uracil content (which iseasily controlled in PCR), rather than time of reaction and size of DNA(which is difficult to control). Size fractionation and purification maybe obviated by the use of Ung-End fragmentation, since the reaction goesto completion, with the average fragment size being a function of uracilcontent only. Note that, as with conventional DNase fragmentation andsize fractionation, Ung-End fragmentation is used for shuffling a singleDNA sequence or family of related DNA sequences. The use of Ung-Endfragmentation along with PCR assembly provides for single-tube DNAshuffling, which can be carried out, e.g., in microtiter plates.

Important considerations in the design of a single-tube shufflingreaction include methods for minimizing carry-over of the plasmidtemplate DNA used to generate uracil-containing DNA for shuffling. Asimple solution is to incorporate uracil into the plasmid template viagrowth in a dut-1 ung-1 double mutant of Escherichia coli, such asstrain CJ236 (Warner et al. (1981) “Synthesis and metabolism ofuracil-containing deoxyribonucleic acid in Escherichia coli” J.Bacteriol. 145(2):687-695; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods Enzmmol.15:367-382) or by PCR. Likewise, incorporation of uracil into primersfor generating uracil-containing DNA minimizes carry-over of primersinto the assembly reaction. Reduction in transformation efficiency ofshuffled product using Ung-End fragmentation can result due to residualuracil. Where this is problematic, transformation of shuffled productsinto an ung mutant of E. coli assists in cloning processes.

Growth of plasmid in a dut-1 ung-1 E. coli mutant (e.g. strain CJ236)for uracil incorporation followed by Ung-End fragmentation and PCRassembly provides a quick, single-tube method of shuffling a wholeplasmid or family of plasmids. Growth of plasmids in an E. coli dut ungstrain bearing a strong mutator allele (e.g. dut ung mutD5) orcombination of mutator alleles for in vivo mutagenesis, as well as,uracil incorporation into plasmid DNA coupled with Und-End fragmentationand PCR assembly is a powerful and simple means of rapidly evolving thefunction of a plasmid. Uracil content of plasmid DNA (and consequentlyaverage fragment size following Ung-End fragmentation) following growthin a dot ung strain is modulated by the addition of exogenous uridine orthymidine. In addition, uracil content is effected using strains bearingalternative dut and/or ung alleles, such as the leaky dut-4 allele forless frequent uracil incorporation (Hays et al. (1981) “Recombination ofuracil-containing Lambda bacteriophages” J. Bacteriol. 145(1):306-320)or be using other alleles which effect cellular dUTP levels or uracilincorporation or removal from DNA. Also, plasmid multimerizationgenerated by Und-End fragmentation and PCR assembly of uracil-containingplasmid can be directly transformed into naturally competent bacteria,such as Bacillus subtilus 168 derivatives, which are more efficientlytransformed by plasmid multimers.

Note that uracil glycosylases and 5′ AP endonucleases are ubiquitous.They have been characterized in both eukaryotic and prokaryotic cells,as well as viruses (Freidberg et al. (1995) DNA Repair and Mutagenesis.pp. 1-698. ASM Press. Washington, D.C.). Many of these can be used forUng-End fragmentation.

In addition to cleaving 5′ to AP sites, AP nucleases (such asExonuclease III, Endonuclease IV, and Endonuclease V) recognize andcleave DNA at sites damaged by oxidizing agents or alkylating agents.Endonculease V additionally cleaves DNA at A/C and A/A mismatches and atdeoxyinosine. Thus, the use of controlled dITP incorporation (e.g.,during oligonucleotide synthesis used in construction of the nucleicacid of interest) and Endonuclease V treatment enables a single enzymemethod for DNA fragmentation. Reagents and bacterial strains for Ung-Endfragmentation can easily be incorporated along with PCR reagents into asimple DNA shuffling kit.

Amplifications with Decreasing Uracil Concentrations:

The following protocol provides an illustrative example of performingamplifications at multiple Uracil concentrations. In an automatedprocess, e.g., in an integrated diversity generation device, appropriateuracil concentrations are optionally calculated, e.g., based onempirical data, to produce a desired fragment length and optimizediversity generation. For example a programmed thermocycler isoptionally used to create appropriate nucleic acids for shuffling, e.g.,having a desired amount of uracil incorporation. The programmedthermocycler can be operably coupled to a fragmentation device thatproduces fragments of a desired length from the uracil containingnucleic acids. The fragments are then used to generate diverse nucleicacids.

First, 50 μl 10 mM dUTP Stock Mixtures are prepared for a dUTPtitration.

100 mM dNTPs stocks are prepared as follows: 10 mM 8 mM 6 mM 4 mM 2 mM 0mM dUTP dUTP dUTP dUTP dUTP dUTP 100 mM 5 5 5 5 5 5 dGTP 100 mM 5 5 5 55 5 dCTP 100 mM 5 5 5 5 5 5 dATP 100 mM 0 1 2 3 4 5 dTTP 100 mM 5 4 3 21 0 dUTP smp H₂O 30 30 30 30 30 30

Second, 100 μl PCR Reactions are made: /100 μl /800 μl smp H20 45 μl 360μl 3.3 X TthXL Buffer 33 264 25 mM MgOAC 10 60 10 mM dNTP Mix 4 32 20pmol/μl Protease Forward 2.5 20 20 pmol/μl Protease Reverse 2.5 20^(˜)100 ng/μl plasmid p3RcCll2 1 6 (XL1-Blue) 2 U/μl TthXL 2 16

Third, Reaction Mixes are prepared with all components except the dNTPMix. 96 μl of Reaction Mix are aliquoted into, e.g., 6 PCR tubes. 4 μlof each of the dNTP Mixes are added to samples of Reaction Mix. Thetubes are placed in a Stratagene RoboCycler using the followingsettings: 1x 2 min@94° C. 30 sec @ 50° C. 1 min @ 72° C. 29x 30 sec @94° C. 30 sec @ 50° C. 1 min @ 72° C.

Finally, 10 μl of each amplification is run on a standard 0.7%Agarose/TBE gel or other separation system.

Enzymatic Treatment with Uracil N-Glycosylase and/or Endonuclease IV:

10 μl of the 0.32, 0.24, 0.16, and 0.0 mM dUTP reactions are aliquotedinto 4 wells of a PCR strip. No enzyme is added to the first aliquot,0.5 μl of 1 U/μl HK™-UNG N-Glycosylase (Epicentre Technologies) to thesecond, 0.5 μl of 2 U/μl E. coli Endonuclease IV (EpicentreTechnologies) is added to the third aliquot, and 0.5 μl of each enzymeis added to the fourth aliquot. The reactions are Incubated for 2 hoursat 37° C. The reactions are then heated for 10 min at 94° C., thenplaced on ice. 10 μl of each reaction are then run on a 1.5% Agarose/TBEgel.

Assembly of Fragments:

Uracil titrations and 100 μl amplifications are repeated to generatemore test DNA. The QIAGEN QIAquick PCR Purification Kit is used toremove primers and unused dNTPs from reactions according to QIAGEN'sinstructions, eluting with 55 μL of smp water. The following is added toall 6 pcr reactions to bring to 100 μl total volume: /100 μl smp water 7μl Reaction in smp water 50 3.3 X TthXL Buffer 33 25 mM MgOAc 10

To 50 μl of each of the 6 reactions, 2.5 μl of 1 U/ml HK™-UNGN-Glycosylase and 2 U/μl E. coli Endonuclease IV is added. The reactionsare incubated for 2 hours at 37° C., then for 10 min at 94° C., and thencooled to 4° C. in a Thermocycler. Untreated reactions are saved foragarose gel analysis. 25 μl of each reaction is removed and saved foragarose gel analysis. To the remaining 25 μl, 25 μl of the followingAssembly Mix is added: /100 μl /200 μl smp water 4 45 μl 90 33 X TthXLBuffer 33 66 25 mM MgOAc 10 20 10 mM dNTP Mix (no Ura) 80 16 2 U/μlTthXL 4 8

The reactions are placed in a Stratagene RoboCycler using the followingsettings: 1x 2 min@94° C. 30 sec @ 50° C. 1 min @.72° C. 29x 30 sec @94° C. 30 sec @ 50° C. 1 mm @ 72° C.

For each uracil concentration, 10 μl of the original PCR reaction isrun, 10 μl of fragments, and 10 μl of assembly reaction on a 1.5%Agarose/TBE gel.

Fragments from the assembly reaction are rescued using PCR with nestedprimers in 100 μl reactions.

Ung-End Fragmentation of E. coli dut ung Grown Plasmid DNA:

Electrocompetent E. coli strain CJ236 (pCJ105 (Cam^(r) F′)/1 dut1 ung1thi-1 relA1) is prepared as follows. Strain CJ236 is Streaked on LB+30μg/ml chloramphenicol and incubated overnight at 37° C. Cells arescraped from the plate into 5 ml LB and inoculated into 250 ml LB to astarting OD₆₀₀ of 0.100. The culture is shaken at 37° C. The culture isplaced on ice for 30 min when at OD₆₀₀ 0.4-0.5 and prepared via standardelectrocompetence procedures, freezing in 220 μl aliquots in 10%Glycerol.

Transformation of strain CJ236 with plasmid is performed as follows. 0.5mg of plasmid are added into 100 ml of electrocompetent strain CJ236 viastandard a electroporation protocol. 10⁻¹ to 10⁴ dilutions are plated onLB+100 μg/μl Ampicillin and incubated overnight at 37° C. Atransformation efficiency of about 2×10⁸ transformants/μg plasmid areobserved. 8 transformants are patched to an LB+Amp100 stock plate andincubated overnight at 37° C. CJ236 in inoculated into 3 ml LBBroth+Amp100, unsupplemented, and supplemented with 500 μg/ml Uradine(to see if fragment size is modulated by supplementation). The culturesare shaken overnight at 37° C. Plasmid DNA is prepared from 1.5 ml withthe aid of a Qiagen Miniprep Spin Kit, suspending plasmid DNA in 50 μlsmp water. A₂₆₀ and A₂₈₀ of a 1:20 dilution in smp water is read andquantitated. Plasmid in CJ236 in LB+Amp100=0.34 μg/μl; plasmid in CJ236in LB+Amp100+Ura500=0.35 μg/μl; plasmid in XL1-Blue in LB+Amp100=0.7μg/μl.

Fragmentation Example: Automated DNA Fragmentation Using DNase-PlasticCo-Polymers

Fragmentation is currently performed by the addition of DNaseI to DNA insolution. This can result in variable fragmentation. For example, PCRproducts are often fragmented less well than plasmids, presumably as aresult of residual salts following purification of the PCR product. Thisexample provides an automated process in which DNA is fragmented andspecific sized fragments are purified, speeding the process greatly.

Immobilized DNase on support resin beads can be used for fragmentation,with DNA to be fragmented passing over a column made of the beads. Thisavoids the problem of salts in the solution which are removed by gelfiltration.

An extension of this procedure is to encapsulate the DNase in apolymeric (plastic) resin. Wang et al. (1997) “Biocatalytic plastics asactive and stable materials for biotransformations” Nat Biotechnol2:15(8):789-93 and the references therein describe Biocatalytic plastictechnology generally. Resin encapsulation has the advantage ofstabilizing the enzyme greatly: no loss of activity is seen even after30 or more days. Synthesis of a stable DNase resin avoids the need tore-calibrate the column to account for the loss of activity. Using afixed initial concentration of DNA, DNA fragment size can be determinedby the flow rate through the column. Fractions can be collectedcontaining known fragment sizes.

Encapsulated DNAse resin can then be used as a component of an automatedDNA shuffling system as set forth herein. That is, fragmentation can beperformed in a flowing fashion, across DNAse or other nuclease columns.This flow-through fragmentation can be performed in an “in line” or“off-line” fashion. For example, the columns can be incorporated intothe fluid handling module(s) herein and performed as part of a fluidtransfer of material to be fragmented (in line fragmentation).Alternately, fragmentation columns can be a separate module in thesystem.

Although described above in terms of columns for purposes ofillustration, it will be appreciated that non-column based methods canutilize particle-bound or encapsulated nucleases, e.g., in a beadpanning or chip-based format.

(5.) Recombination/Resynthesis/Amplification Module

The recombination resynthesis module permits hybridization ofcomplementary (or partially complementary) nucleic acids, followed byPCR-based resynthesis of hybridized nucleic acids, typically usingmultiple cycles of PCR (a variety of PCR-based re-synthesis methods,including staggered extension process (“StEP”) PCR are set forth in thereferences above), or ligation (e.g., via LCR). In general, PCR can beused to “sew” sets of overlapping nucleic acids together, simply byperforming multiple cycles of PCR on overlapping nucleic acid fragments.Similarly, ligases can be used to ligate overlapping (or evennon-overlapping) nucleic acid fragments (with or without apolynucleotide extension (e.g. polymerase-mediated) step between cyclesof ligation). Where PCR is used, the recombination/resynthesis modulealso optionally performs nucleic acid amplification, i.e., by PCR.

The amplification of arrays and duplicate arrays is also an importantfeature of the invention, as this amplification provides material forsubsequent operations (2^(nd) round diversity generation reactions suchas shuffling, cloning, sequencing, etc.). For example a duplicateamplified array can be formed by copying a master array, or a portionthereof, and generating amplicons of the members of the resultingduplicate array to form an amplified array of nucleic acids. Anyavailable amplification methods can be used, including amplifyingnucleic acids in physical or logical arrays by PCR, LCR, SDA, NASBA,TMA, Q∃-replicase amplification, etc.

Common physical elements for the resynthesis module include heating andoptionally cooling elements to perform PCR, containers to hold nucleicacids to be resynthesized (microtiter trays, chips, test tubes, etc.).For example, standard PCR thermocyclers can be incorporated into thismodule, i.e., in combination with appropriate instruction sets toperform synthesis recombination and amplification. For example, a set ofinstructions is optionally embodied in a programmed thermocycler, acomputer operably coupled to a thermocycler, or in a web page that canbe used to instruct a thermocycler. The set of instructions typicallyreceives user input data and sets up cycles to be performed on thethermocycler, e.g., a programmed thermocycler. The user input datatypically includes one or more parental nucleic acid sequence, a desiredcrossover frequency, an extension temperature, and/or an annealingtemperature, and the like. From such user input data, a set ofinstructions, e.g., embodied in a computer readable medium, creates acycle which is performed by the programmed thermocycler. For example, aset of instructions optionally sets up a cycle to amplify one or moreparental nucleic acid sequence and fragment the one or more parentalnucleic acid sequence to produce one or more nucleic acid fragment. Insome embodiments, the cycle is programmed or instructed to pause beforefragmenting to allow the addition of fragmentation enzymes, e.g., tofragment nucleic acids that have had uracil residues incorporatedtherein. The fragments are then reassembled to produce one or moreshuffled nucleic acid; which is optionally amplified, all according tothe set of instructions or calculations.

Amplifiers typically include some sort of heating element and can alsoinclude a cooling element. Such elements commonly include (but are notlimited to) resistive elements, programmable resistors, micromachinedzone heating chemical amplifiers, Peltier solid state heat pumps, heatpumps, resistive heaters, refrigeration units, heat sinks, JouleThompson cooling devices, a heat exchanger, a hot air blower, etc. Anyof the above elements are optionally operably coupled to a computercomprising a set of instructions which directs or instructs the elementsin the amplification process, e.g., according to user input data orcomputer calculated predictions.

Recently, attempts have been made to shorten the time required for eachcycle of PCR, an advantage in the present method, in that reduction inthis time increases the overall throughput of the system. Such methodsoften reduce the time by, for example, performing the PCR in devicesthat allow rapid temperature changes. The use of apparatus that allowgreater heat transfer, e.g., incorporating thin-walled tubes, turbulentair-based machines, and the like also facilitate the use of shortercycle times. For example, the RapidCycler™, from Idaho Technologies,Inc. (Salt Lake City, Utah) allows relatively rapid ramping timesbetween each temperature of a PCR and relatively efficient thermaltransfer from the cycler to the samples. Similarly, the RAPID(Ruggedized Advanced Pathogen Identification Device) from IdahoTechnologies, Inc. provides a thermal cycler with concurrentfluorescence monitoring to speed analysis as well.

As an alternative or adjunct to standard PCR thermocyclic elements,chip-based PCR can also be incorporated into the present invention. Arecent example of chip-based PCR was discussed by Kopp et al. (1998)“Chemical Amplification: Continuous Flow PCR on a Chip” Science280:1046-1047. Kopp et al. describe a microfluidic continuous flow PCRsystem where the PCR reactants were flowed through a chip having threediscrete temperature zones. The reagents within the channel underwentessentially instantaneous changes in temperature. Thus, the cycle timein this system reflected the time at each temperature, with nosubstantial temporal contribution from ramping times.

Additional chip-based PCR methods are set forth in U.S. Pat. No.5,587,128 to Wilding et al. Dec. 24, 1996 “MESOSCALE POLYNUCLEOTIDEAMPLIFICATION DEVICES”) which similarly incorporate hot zones and fluidflow to achieve temperature cycling. PCR can also be performed by fluidresistance heating in microchips. For example, U.S. Pat. No. 5,965,410,to Chow, et al., Oct. 12, 1999, “ELECTRICAL CURRENT FOR CONTROLLINGFLUID PARAMETERS IN MICROCHANNELS” describe such devices.

In certain embodiments, non-thermocyclic polymerase mediatedamplification can be achieved, i.e., using a chemical denaturationdevice or an electrostatic denaturation device. For example U.S. Pat.No. 5,939,291 by Loewy et al., Aug. 17, 1999 “MICROFLUIDIC METHOD FORNUCLEIC ACID AMPLIFICATION” describes such devices. This invention canalso be used with polymerases capable of performing under unusual orbiochemically challenging environments such as are created under extremeshear forces, temperatures, salt concentrations, or the presence of oneor more non-aqueous solvents and other chemicals. Such enzymes may begenerated via the shuffling and mutagenesis techniques disclosed hereand elsewhere in the art.

(6.). PCR Amplification of Individual Fragments

It is generally preferable to amplify diversified nucleic acids by PCRor any of the other amplification techniques herein prior to an in vitrotranscription and translation step. This is desirable because singlecopy genes can become damaged or otherwise compromised during the courseof the transcription/translation or assay steps, making rescue of thegenetic material problematic. Also, PCR amplification of a single genecopy can be suboptimal, although it is known to be possible (Ohuchi etal. (1998) “In vitro Generation of protein libraries using PCRamplification of a single DNA molecule and coupled proteintranscription/translation,” Nucleic Acids Res. 26(19):4339-4346). Thetrue number of starting genes in each reaction can be estimated usingquantitative PCR. Such quantification involves, e.g., imaging of theamplified products via methods involving fluorescence detection,fluorescence resonance energy transfer, autoradiography,chemilumniescence or visible dyes.

(7.) Measuring Diversity/Library Quality Module

The diversity generation module can include a nucleic acid deconvolutionmodule (or this module can exist separately to identify nucleic acids inother portions of the system). For example, the diversity generationmodule can include an identification portion, which identifies one ormore nucleic acid portion or subportion.

A variety of nucleic acid deconvolution methods can be used, includingnucleic acid sequencing, restriction enzyme digestion, dye incorporationand the like. The module can determine a recombination frequency (e.g.,by dye incorporation, labeled nucleotide incorporation, sequencing,restriction enzyme digestion, rescue PCR, etc.) or a length of product(by any molecular sizing technology, or by dye incorporation, nucleotideincorporation, sequencing, restriction enzyme digestion, rescue PCR,etc.), or both a recombination frequency and a length, for the resultingelongated nucleic acids. Detection can be by detecting labels associatedwith nucleic acid products (e.g., detection of a dye, radioactive label,biotin, digoxin, a fluorophore, etc.), or simply by detecting thenucleic acid directly. Secondary assays such as fluorogenic 5′ nucleaseassays can be used for detection. For example, the extent of PCRamplification can be determined by incorporation of a label into one ormore amplified elongated nucleic acid, a fluorogenic 5′ nuclease assay,TaqMan, FRET, etc.

In general, an important factor in producing diverse nucleic acids inthe diversity generation module(s) is the ability to measure thediversity which is generated. For example, if there is limitedrecombination in a shuffling reaction, the library of nucleic acidswhich is produced is often not sufficiently diverse for optimalscreening of an activity of interest. Thus, in preferred embodiments,the shuffling module assesses the degree of diversity, generally beforeany screening is performed.

Diversity assessment can be performed in a number of ways. Aliquots ofdiverse populations of nucleic acids can be cloned or amplified (e.g.,via standard primers which provide for amplification of all or leastsome members of the pool) by limiting dilution. These nucleic acids canthen be sequenced, e.g., using automated sequencing methods andapparatus. The diversity of the population is then assessed, e.g., usingsequence alignment algorithms, by visual inspection, or the like. Poolswhich are determined to be diverse can then be selected for activity ofinterest, used as substrates in additional recombination reactions, orthe like.

Sometimes it is possible to make a determination, or an approximation,of diversity without having to sequence members of the population ofnucleic acids. For example, a rescue PCR or LCR reaction can beperformed that is designed to preferentially amplify recombined nucleicacids. In such rescue reactions, rescue PCR or LCR primers are providedwhich correspond to a subset (and, occasionally, only one) of theoriginal parental nucleic acids that were acquired as noted above. Byperforming combinatorial PCR or LCR reactions using such primers, it ispossible to determine whether recombination has taken place between twoor more parental nucleic acids. That is, nucleic acids which areproduced are optionally only amplified in the rescue PCR or LCR processif they have sequences corresponding to two or more parental nucleicacids (excluding PCR/LCR control reactions). Recombination events aredetected for using appropriate combination of primers in the rescuereaction.

PCR/LCR products can be detected in solution, eliminating the need forseparation or sequencing (although these approaches can be used, ifdesired, to provide more complete information of what sequences arerescued). For example, the amount of double-stranded DNA in the rescuedpool provides an indication as to whether a PCR/LCR was successful.Thus, If there is double-stranded DNA following a rescue PCR/LCRamplification on a subset of the pool, then it is likely that theassembly reaction worked properly, producing recombinant nucleic acids.Simply monitoring double-strand DNA specific dye incorporation in aPCR/LCR rescue reaction provides at least a first approximation of theefficiency of the fragmentation and reassembly process.

For example, the PicoGreen dsDNA quantitation reagent (available e.g.,from Molecular Probes) can be used to monitor and quantitate dsDNA.Similarly, the OliGreen ssDNA reagent can be used to monitor andquantitate ssDNA (including oligonucleotides) and the RiboGreen RNAquantitation reagent can be used to monitor RNA. See, e.g., Haugland(1996) Handbook of Fluorescent Probes and Research Chemicals SixthEdition by Molecular Probes, Inc. (Eugene Oreg.) andhttp://www.probes.com/handbook (the on-line 1999 version of the Handbookof Fluorescent Probes and Research Chemicals Sixth Edition by MolecularProbes, Inc.) (Molecular Probes, 1999). For example, Molecular Probes1999, Chapter 8 (e.g., section 8.2) provides details regardingquantitation of DNA in solution.

The PicoGreen reagent (e.g., Molecular Probes Nos. P-7581, P-11495) andKit (Molecular Probes Nos. P-7589, P-11496) accurately quantitate aslittle as 25 pg/mL of double-stranded DNA (dsDNA) in a fluorometer or250 pg/mL (typically 50 pg in a 200 μL volume) in a fluorescencemicroplate reader. The PicoGreen assay is greater than 10,000 times moresensitive than conventional UV absorbance measurements at 260 nm (anA260 of 0.1 corresponds to a 5 μg/mL dsDNA solution). Although thePicoGreen reagent is not actually specific for dsDNA, it showsa >1000-fold fluorescence enhancement upon binding to dsDNA, and lessfluorescence enhancement upon binding to single-stranded DNA (ssDNA) orRNA, making it possible to quantitate dsDNA in the presence of ssDNA,RNA, proteins or other materials. Thus, the PicoGreen reagent allowsdirect quantitation of PCR amplicons without purification from thereaction mixture and makes it possible to detect low levels of DNAcontamination in recombinant protein products.

Protocols for the PicoGreen assay are amenable to high-throughputscreening in the systems herein. The dye is added to the sample (e.g.,in a microtiter tray) and incubated for about five minutes, and then thefluorescence is measured. In addition, the fluorescence signal frombinding of the PicoGreen reagent to dsDNA is linear over at least fourorders of magnitude with a single dye concentration. Linearity ismaintained in the presence of several compounds commonly found innucleic acid preparations, including salts, urea, ethanol, chloroform,detergents, proteins and agarose.

For detecting oligonucleotides and other ssDNA the OliGreen ssDNAquantitation reagent from Molecular Probes (No. O-7582) and/or (No.O-11492) can be used). The OliGreen ssDNA quantitation reagent enablesquantitatation of as little as 100 pg/mL of ssDNA—200 pg in a 2 mL assayvolume with a standard fluorometer or 200 pg in a 200 μL assay volumeusing a fluorescence microplate reader. Thus, quantitation with theOliGreen reagent is about 10,000 times more sensitive than quantitationwith UV absorbance methods and at least 500 times more sensitive (andfar faster, with a greater throughput) than detecting oligonucleotideson electrophoretic gels stained with ethidium bromide.

The OliGreen ssDNA quantitation reagent does exhibit fluorescenceenhancement when bound to dsDNA and RNA. Like the PicoGreen assay, thelinear detection range of the OliGreen assay in a standard fluorometerextends over four orders of magnitude—from 100 pg/mL to 1 μg/mL—with asingle dye concentration. The linearity of the OliGreen assay is alsomaintained in the presence of several compounds commonly found tocontaminate nucleic acid preparations, including salts, urea, ethanol,chloroform, detergents, proteins, ATP and agarose (see, e.g., theOliGreen product information sheet from Molecular Probes); however, manyof these compounds do affect signal intensity, so standard curves aretypically generated using solutions that closely mimic those of thesamples. The OliGreen reagent shows a large fluorescence enhancementwhen bound to poly(dT) but only a relatively small fluorescenceenhancement when bound to poly(dG) and little signal with poly(dA) andpoly(dC). Thus, it is helpful to use an oligonucleotide with similarbase composition when generating a standard curve for concentrationdependence. The OliGreen ssDNA quantitation reagent can be used forquantitation of antisense oligonucleotides, aptamers, genomic DNAisolated under denaturing conditions, LCR/PCR primers, phosphorothioateand phosphodiester oligodeoxynucleotides, sequencing primers,single-stranded phage DNA, etc.

Other dyes such as the Cyanine Dyes and Phenanthridine Dyes can also beused for Nucleic Acid Quantitation in Solution and are, therefore,adaptable to use in the present invention. See, Molecular Probes, Supra,for a discussion of these and many other nucleic acid staining andquantitation dyes.

In one embodiment, a real time PCR assay system such as the “TaqMan”system is used for library quality determinations. Real time PCR productanalysis by, e.g., FRET or TaqMan (and related real timereverse-transcription PCR) is a known technique for real time PCRmonitoring that has been used in a variety of contexts (see, Laurendeauet al. (1999) “TaqMan PCR-based gene dosage assay for predictive testingin individuals from a cancer family with INK4 locus haploinsufficiency”Clin Chem 45(7):982-6; Laurendeau et al. (1999) “Quantitation of MYCgene expression in sporadic breast tumors with a real-time reversetranscription-PCR assay” Clin Chem 59(12):2759-65; and Kreuzer et al.(1999) “LightCycler technology for the quantitation of bcr/abl fusiontranscripts” Cancer Research 59(13):3171-4. Examples of theseembodiments are set forth in more detail in the two following examples.

Example: Parallel Determination of Family Library Quality WithoutCloning or Sequencing

A significant rate limiting step in the creation of a shuffled librariesis the determination of library quality. Since chimera formation dependson multiple parameters (fragment size, gene size, GC content, annealingtemperature, extension temperature, number of parents, homology betweenparents) it is difficult to predict the conditions required for acertain crossover frequency.

An alternative to complete control of the shuffling process is to gainprecise control (i.e. for reproducibility) over important parameters(such as fragment size, annealing and extension temperatures, parentalrepresentation etc) and then to make multiple libraries in which theseare systematically varied, e.g., in a microtitre plate format. Theproblem then is how to assess rapidly the quality of these librarieswithout the labor-intensive and costly processes of cloning andsequencing.

There are two common determinants of shuffled libraries: the frequencyof recombination used to produce the library members, and the frequencywith which frame shifts or deletions prevent the synthesis offull-length protein.

The TaqMan system (Perken Elmer Biosystems) provides one example ofavailable technology that can be adapted to address these problems.TaqMan is a real-time PCR detection system that works as follows. Twooligonucleotides are used as amplification primers, e.g., about two orthree hundred bases apart. A third primer, complementary to a section ofDNA between these primers, is labeled with a fluorescent dye and afluorescence quencher. During PCR, the third oligonucleotide anneals tothe single stranded product DNA, and is then degraded by the 5′ to 3′exonuclease activity of the polymerase as it extends through the regionto which the labeled oligonucleotide is annealed. Degradation of thelabeled oligonucleotide separates the fluorescent dye from the quencher,resulting in an increase in fluorescence. The cycle number at which anincrease in fluorescence appears indicates the abundance of a particulartemplate.

The TaqMan system can be adapted to measure the abundance of variouschimeras in a microtiter format. Varying the primers and indicatoroligonucleotides used allows detection of different classes of chimeras(see, FIG. 9). A simple tiered screen can used in which libraries arefirst screened for the presence of a fragment of B or C, incorporatedbetween two fragments of A. Libraries that score well in this test couldthen be tested for more complex chimera arrangements. Finally the bestfew (5 or so) libraries are cloned into a translational-coupling vector,and full-length variants are picked, screened and sequenced. This, inturn, generates feedback about the types of chimeras that are the bestindicators for a specific function, and the relationship between thesimple chimera indicator described here and the real sequencesgenerated.

As shown in FIG. 9, a labeled B oligo can be used to measure therelative differences of, e.g., 8 possible crossovers. Alternately,several different fluorescently labeled oligos can be used in the samewell of a reaction tray or other container. In this scheme, a library istested by amplifying with a specific primer and fluorescence of A, B andC for different indicator dyes are measured as a function of the numberof cycles (e.g., PCR cycles). This gives an indication of the frequencyof the types of crossovers present in the library sample, illustratedschematically.

This kind of library screening dramatically increases the throughput forlibrary assessment as compared to previous methods.

An alternative to TaqMan is the use of molecular beacons to assesslibrary quality. Molecular beacons are oligonucleotide probes that canreport the presence of specific nucleic acids in homogeneous solutions(Tyagi and Kramer (1996) “Molecular beacons: probes that fluoresce uponhybridization.” Nat Biotechnol 14, 303-308. They are used for real-timemonitoring of PCR or other amplification reactions and for the detectionof RNAs within living cells. Molecular beacons are hairpin-shapedmolecules with an internally quenched fluorophore whose fluorescence isrestored when they bind to a target nucleic acid (see Tyagi and Kramer,id). They are designed so that the loop portion of the molecule is aprobe sequence complementary to a target nucleic acid molecule. The stemis formed by an annealing of complementary arm sequences on the ends ofthe probe sequence. A fluorescent moiety is attached to the end of onearm and a quenching moiety is attached to the end of the other arm. Thestem keeps these two moieties in close proximity to each other, causingthe fluorescence of the fluorophore to be quenched by energy transfer.When the probe encounters a target molecule, it forms a hybrid that islonger and more stable than the stem hybrid and its rigidity and lengthpreclude the simultaneous existence of the stem hybrid. Thus, themolecular beacon undergoes a spontaneous conformational reorganizationthat forces the stem apart, and causes the fluorophore and the quencherto move away from each other, leading to the restoration of fluorescencewhich can be detected. Further details on Molecular Beacons and theiruse can be found in the following references: Tyagi et al. (1998)“Multicolor molecular beacons for allele discrimination” Nat Biotechnol16:49-53; Matuso (1998) “In situ visualization of mRNA for basicfibroblast growth factor in living cells” Biochimica Biophysica Acta1379:178-184; Sokol et al. (1998) “Real time detection of DNA-RNAhybridization in living cells” Proc Natl Acad Sci USA 95:11538-11543;Leone et al. (1998) “Molecular beacon probes combined with amplificationby NASBA enable homogeneous, real-time detection of RNA” Nucleic AcidsRes 26:2150-2155; Piatek et al. (1998) “Molecular beacon sequenceanalysis for detecting drug resistance in Mycobacterium tuberculosis”Nat Biotechnol 16:359-363; Kostrikis et al. (1998) “Spectral genotypingof human alleles” Science 279:1228-1229; Giesendorf et al. (1998)“Molecular beacon: a new approach for semiautomated mutation analysis”Clin Chem 44:482-486; Marras et al. (1999) “Multiplex detection ofsingle-nucleotide variations using molecular beacons” Genet Anal14:151-156; and Vet at al. (1999) “Multiplex detection of fourpathogenic retroviruses using molecular beacons” Proc Natl Acad Sci USA96:6394-6399.

Thus, the presence or absence of any specific nucleic acid (includingany mutated nucleic acid) can be monitored in real time via the use ofMolecular Beacons.

Example: Monitoring of Recombination using Fluorescence Energy Transfer

After performing a diversity generation reaction, an extensive analysisof the library can be performed to check whether there was recombinationbetween genes (or other nucleic acids) and at what frequency. Animmediate answer to those question speeds up the construction of therelevant libraries. Furthermore, if the monitoring is continuous duringthe shuffling reaction, the conditions can be changed to optimizerecombination, even before the end of the reaction.

The process in this example utilizes real time PCR analysis based uponFRET. The method uses “light cycler” techniques (De Silva et al (1998)Biochemica “Rapid Genotyping and Quantification with HybridizationProbes Rapid Genotyping and Quantification on the LightCycler withHybridisation Probes” 2:12-15, and De Silva et al (1998) Biochemica “TheLightCycler—The Smartest Innovation for More Efficient PCR” Biochemica2: 4-7).

Fluorescent resonance energy transfer (FRET) is a distance dependentexcited state interaction in which emission of one fluorophore iscoupled to the excitation of another which is in proximity (close enoughfor an observable change in emissions to occur). Some excitedfluorophores interact to form excimers, which are excited state dimersthat exhibit altered emission spectra (e.g., phospholipid analogs withpyrene sn-2 acyl chains); see, Haugland (1996) Handbook of FluorescentProbes and Research Chemicals, Published by Molecular Probes, Inc.,Eugene, Oreg., e.g., at chapter 13).

The Forster radius (R_(o)) is the distance between fluorescent pairs atwhich energy transfer is 50% efficient (i.e., at which 50% of exciteddonors are deactivated by FRET. The magnitude of R_(o) is dependent onthe spectral properties of donor and acceptor dyes:R_(o)=[(8.8×10²³)(K²)(n⁻⁴)(QY_(D))(J)(S)]^(1/6) Å, where: K²=dipoleorientation range factor (range 0 to 4, K²=⅔ for randomly orienteddonors and acceptors); QY_(D)=fluorescence quantum yield of the donor inthe absence of the acceptor; n=refractive index; and, J(S)=spectraloverlap integral=IM_(A)(S)·F_(D)S·S⁴dScm³M⁻¹, Where M_(A)=extinctioncoefficient of acceptor and F_(D)=Fluorescence emission intensity ofdonor as a fraction of total integrated intensity. Typicaldonor-acceptor pairs include fluorescein/Cy5,fluorescein/tetramethylrhodamine, IAEDANS/fluorescein,Fluorescein/Fluorescein, BODIPY/BODIPY and EDANS/DABCYL. An extensivecompilation of R_(o) values are found in the literature; see, Haugland(1996) Handbook of Fluorescent Probes and Research Chemicals Publishedby Molecular Probes, Inc., Eugene, Oreg. at page 46 and the referencescited therein.

In brief, two probes are labeled with different fluorophores. The twoprobes are complementary to a specific region of a gene to be analyzed.If the desired genotype (recombination event) is present in the sample,the probes bring two fluorophores into close proximity (e.g., withinR_(o)), allowing a transfer of energy between them. This transfer ofenergy can be monitored using a device such as the one described in theDe Silva et al. references (id); see also, the LightCycler fromAmersham.

This approach can be used in shuffling or other diversity generatingreactions using automated techniques. In order to label the DNAmolecules, constructed, e.g., during PCR or LCR reactions, nucleotideslabeled with fluorophores are used and are introduced by the DNApolymerase or other enzymes into the molecule, or via automatedsynthetic approaches. The fluorophores are excited and detected bysystem.

For example, two genes to be shuffled can be labeled using this method,e.g., one with fluorescein, and the other with Cy5 in a PCR reaction(both fluorophores are available, e.g., from Amersham Pharmacia). Thelabeled genes are fragmented, e.g., using DNaseI before being shuffledby the system. Recombination between the two genes brings thefluorescein molecule next to the Cy5 molecule, and, e.g., after eachcycle the system excites the fluorescein. The fluorescein then transfersits energy either to the Cy5 molecule, if it is proximal, or to themedia if it is not. The system then detects light at the wavelength ofemission of Cy5, providing an indication of FRET. Similarly, FRET can beused to assess recombination frequency by solution-phase or solid-phasehybridization to differentially labeled fluorescence-coupledoligonucleotide, PCR amplified or restriction fragment-generated probes.

(8.) Non-Coding Control Sequences

Quite commonly, output nucleic acids from the shuffling or mutagenesismodule comprise one or more sequences which control transcription ortranslation or which facilitate downstream processing of the nucleicacid (e.g., cloning). These sequences include promoters, enhancers,ribosome binding sites, translation initiation regions, transcriptioninitiation regions, universal PCR primer binding sites, sequencingprimer binding sites, restriction enzyme digestion sequences and othersequences of known activity. Ausubel, Sambrook, Berger and a number ofother references herein provide an introduction to sequences useful ingenetic engineering. Many such sequences are known and can easily beprovided in the present methods, if desired. For example, including suchsequences as part of PCR or ligase-directed gene synthesis is aconvenient way of incorporating such sequences of interest.

Amplifying recombinant nucleic acids in physical or logical arrays, oramplifying elongated nucleic acids in master arrays, duplicate arrays orother arrays herein can include, as a feature of the amplification, theincorporation of one or more transcription or translation controlsubsequence into the elongated nucleic acids, recombinant nucleic acidsin the physical or logical array, intermediate nucleic acids producedusing elongated nucleic acids or recombinant nucleic acids in thephysical or logical array as a template, partial or complete copies ofelongated nucleic acids or recombinant nucleic acids in the physical orlogical arrays, and the like. One or more transcription or translationcontrol subsequence can be ligated to the elongated nucleic acids, therecombinant nucleic acids in the physical or logical array, intermediatenucleic acids produced using the elongated nucleic acids or therecombinant nucleic acids in the physical or logical array as atemplate, partial or complete copies of the elongated nucleic acids orthe recombinant nucleic acids in the physical or logical array, etc. Forexample, the one or more transcription or translation controlsubsequences can be hybridized or partially hybridized to the abovenucleic acids during any nucleic acid amplification or polymerase orligase mediated method herein.

(9.) Isolation of Single DNA Molecules from a Mixed Pool WithoutBacterial Transformation

This section describes a method that allows pieces of DNA to be singlyisolated from a pool and amplified for sequencing or other process(e.g., shuffling or in vitro translation) without the use of a hostorganism. The method is both faster and more reliable than traditionalcloning. The method is based upon the ability to form particles fromindividual pieces of DNA that can then be isolated and dispensed intoindividual wells. The particles are degraded and each piece of DNA isamplified to give enough material for sequencing or other downstreamoperations.

The advantage of this protocol is that the particles are formed due tothe physical nature of the DNA polymer, and as such, the protocol issequence and context independent. Thus all pieces of DNA haveapproximately the same chance of being amplified at the end of theprocess, unlike traditional cloning methods.

DNA Library Preparation

When cloning from genomic DNA, the DNA is usually cleaved to suitablesize by nuclease (e.g., restriction enzyme) or mechanical treatment. Toamplify the DNA, the ends of each fragment are compatible, e.g., for PCRamplification using standard primers. This is true if the DNA moleculeshave a standard construction with fixed 5′ and 3′ ends (as is usual forRNA or DNA selection constructs and for expression constructs). Forcloning of fragments of unknown DNA (or following mechanical or randomcleavage procedures), this is achieved by ligation of standard primersto the end of each fragment for subsequent ligation into a vector.Fluorescent or other tags can be added to the extension to aid handlingand analysis. Successfully ligated molecules can be enriched in the poolby PCR and purified, if necessary, by standard methods.

Monomolecular Particle Formation

DNA is a rigid polyanionic linear polymer that exists as a monomer insolution with a large radius of gyration as it floats in a random coilstructure. The addition of a polycationic polymer to a solution of DNAcauses the DNA to associate with the polycation and condense in acooperative electrostatic process to yield a compact complex. Due to theelectrostatic nature of the process, there is a tendency for multiplecopies of the two polymers to associate to give large poorly definedmixtures of particles.

Complexation of DNA with single chain cationic detergents is known toform small monomolecular particles (J. Am. Chem. Soc. 1995, 117,2401-2408), but these complexes are unstable to reduction of thedetergent concentration. The ability of single chain detergents to formcomplex is based upon the formation of the polycation at the DNA in atemplate-assisted assembly. Hence addition of such a detergent to asolution of DNA leads to formation of small (˜20 nm) complexes which canthen be dispensed into individual wells. Dilution of the particles witha PCR mix leads to dissolution of the complex, releasing free DNA readyfor amplification.

Complexes formed with detergent can be relatively unstable. However,other methods of forming monomolecular complexes are available. See,e.g., Blessing (1998) Proc. Natl. Acad. Sci. USA 95:1427-1431. In thisprotocol, the single chain cationic detergent contains a chemical moietysuch as a thiol group. Once the complex has formed, the detergents aredimerized (by oxidation for thiols) which yields a stable particle. Oncethe particles are dispensed, the dimerization is reversed (reduction ofthe disulfide) and the complex degrades to yield free DNA. Addition oflipophilic fluorophores to these complexes leads to production of afluorescent particle. This can be used to track the complexes forsorting as described below.

Dispensing the Particles

The charged complexes formed by the protocols outlined above are readilysorted by electrophoretic mobility to remove uncomplexed material.Dispensing these particles into separate wells of a microtiter plateuses, e.g., electrophoresis, e.g., in which the particles travel down acapillary (or channel) in single file, much like in a FACS machine (orchip). A fluorescent detector (e.g., LIF, confocal laser with suitablePMT/CCD) set up at the end of the system detects passage of particlesand directs particles into the receiving well. Flow cytometry systemswhich will sort into microtiter plates of any format, are available,e.g., from Cytomation (Fort Collins, Colo.).

Release of the Free DNA

Stability of the DNA-detergent complex is sensitive to reduction indetergent concentration. Thus, dilution of the particles into a PCR mixleads to dissolution of the complex, releasing free DNA foramplification. The PCR product can then be used for the desired purpose(sequencing, in vitro transcription/translation, etc.).

(10.) Array Copy Systems

During operation of the devices of the invention, populations of nucleicacids can be arranged into one or more physical or logical recombinantnucleic acid arrays. In several of the procedures herein, a duplicate ofat least one of the one or more physical or logical recombinant nucleicacid arrays is produced in the process of amplifying, sequencing, orexpressing members of the nucleic acid array. Thus, in one typicalembodiment, the system includes a shuffled nucleic acid master arraywhich physically or logically corresponds to positions of the shufflednucleic acids in the reaction mixture array. This master array can beaccessed as necessary, e.g., where access of reaction mixture or otherduplicated nucleic acid arrays is not feasible. See also, FIG. 1 b.

In general, the diversity generation module can copy arrays (i.e., themodule can include an array copy function) to produce duplicate arrays,master arrays, amplified arrays and the like, e.g., where any operationis contemplated which could make recovery of nucleic acids from anoriginal array problematic (e.g. where a process to be performeddestroys the original nucleic acids, e.g., recombination methods thatchange the nature of product nucleic acids as compared to startingnucleic acids), or where an elevated stability for the array would behelpful (e.g., where an amplified array can be produced to stabilizeaccessible copies of nucleic acids), or where a normalization ofcomponents (e.g., to provide similar concentrations of reactants orproducts) is useful for recombination, expression or analysis purposes.Copies can be made from master arrays, reaction mixture arrays or anyduplicates thereof.

For example, the diversity generation module optionally dispensesnucleic acids into one or more master multiwell plates and, typically,amplifies the resulting master array of elongated nucleic acids (e.g.,by PCR) to produce an amplified array of elongated nucleic acids. Theshuffling module can include an array copy system which transfersaliquots from the wells of the one or more master multiwell plates toone or more copy multiwell plates.

The array of reaction mixtures can be formed, e.g., by separate orsimultaneous addition of an in vitro transcription reagent and an invitro translation reagent to one or more copy multiwell plates (or otherspatially organizing set of containers), or to a duplicate set thereof,to diversified nucleic acids.

In addition to adding reaction mixture components directly to arrays,reaction mixture components are commonly added to duplicate arrays ofshuffled or otherwise diversified nucleic acids. For example, thereaction mixtures can be produced by adding in vitrotranscription/translation reactants to a duplicate nucleic acid array,which is duplicated from a master array of the shuffled nucleic acidsproduced by spatially or logically separating members of a population ofthe shuffled nucleic acids.

Arraying techniques for producing both master and duplicate arrays frompopulations of shuffled or otherwise diversified nucleic acids caninvolve any of a variety of methods. For example, when forming solidphase arrays (e.g., as a copy of a liquid phase array, or as an originalarray), members of the population can by lyophilized or baked on a solidsurface to form a solid phase array, or chemically coupled or printed(e.g., using ink-jet printing methods) to the solid surface. Similarly,population members can be converted from solid phase to liquid phase byrehydrating members of the population, or by cleaving chemically coupledmembers of the population of shuffled nucleic acids from the solidsurface to form a liquid phase array. One or more physically separatedlogical or physical array members can be accessed from one or moresources of shuffled or otherwise diversified nucleic acids and moved toone or more array destination site (e.g., by pipetting into microtitertrays), where the one or more destinations constitute a logical array ofthe shuffled nucleic acids.

Individual members of an array can be copied in a number of ways. Forexample, members can be amplified and aliquots removed and placed in aduplicate array. Alternately, where the sequences of array members aredeconvoluted (e.g., sequenced) copies can be produced synthetically andplaced into copy arrays. Two preferred ways of copying array members areto use a polymerase (e.g., in amplification or transcription formats) orto use an in vitro nucleic acid synthesizer for copying operations.Typically, a fluid handling system will deposit copied array members indestination locations, although non-fluid based member transport (e.g.,transfer in a solid or gaseous phase) can also be performed.

B. In Vitro Transcription/Translation

In one preferred embodiment of the invention, libraries of nucleic acidsproduced by the various diversity generation methods set forth herein(shuffling, mutation, etc.) are transcribed (i.e., where the diversenucleic acids are DNAs) into RNA and translated into proteins, which arescreened by any appropriate assay. Common in vitro transcription and/ortranslation reagents include reticulocyte lysates (e.g., rabbitreticulocyte lysates) wheat germ in vitro translation (IVT) mixtures, E.coli lysates, canine microsome systems, HeLa nuclear extracts, the “invitro transcription component,” (see, e.g., Promega technical bulletin123), SP6 polymerase, T3 polymerase, T7 RNA polymerase (e.g., Promega #TM045), the “coupled in vitro transcription/translation system” (ProgenSingle Tube Protein System 3) and many others. Many of translationsystems are described, e.g., in Ausubel, supra. as well as in thereferences below, and many transcription/translation systems arecommercially available.

Methods of processing (transcribing and/or translating) diversifiednucleic acids (shuffled, mutagenized, etc.) are provided. In themethods, a physical or logical array of reaction mixtures is provided,in which a plurality of the reaction mixtures include one or more memberof a first population of nucleic acids (including shuffled, mutagenizedor otherwise diversified nucleic acids). A plurality of the plurality ofreaction mixtures further comprise an in vitro transcription ortranslation reactant. One or more in vitro translation products producedby a plurality of members of the physical or logical array of reactionmixtures is then detected. The physical or logical array or reactionmixtures produced by these methods are also a feature of the invention.

Generally, cell-free transcription/translation systems can be employedto produce polypeptides from solid or liquid phase arrays of DNAs orRNAs as provided by the present invention. Severaltranscription/translation systems are commercially available and can beadapted to the present invention by the appropriate addition oftranscription and or translation reagents to arrays of diversifiednucleic acids, e.g., produced by shuffling target nucleic acids andarraying the resulting nucleic acids. A general guide to in vitrotranscription and translation protocols is found in Tymms (1995) Invitro Transcription and Translation Protocols: Methods in MolecularBiology Volume 37, Garland Publishing, NY. Any of the reagents used inthese systems can be flowed or otherwise directed into contact withnucleic acid array members.

Typically, in the present invention, in vitro transcription and/ortranslation reagents are added to an array (or duplicate thereof) thatembodies the diverse populations of nucleic acids generated by diversitygenerating procedures. For example, where the nucleic acids of interestare plated on microtiter trays, the in vitro transcription/translationreagents are added to the wells of the trays to form arrays of reactionmixtures that individually comprise the in vitrotranscription/translation reagents, the nucleic acids of interest andany other reagents of interest.

Several in vitro transcription and translation systems are well knownand described in Tymms (1995), id. For example, an untreatedreticulocyte lysate is commonly isolated from rabbits after treatment ofthe rabbits with acetylphenylhydrazine as a cell-free in vitrotranslation system. Similarly, coupled transcription/translation systemsoften utilize an E. coli S30 extract. See also, the Ambion 1999 ProductCatalogue from Ambion, Inc (Austin Tex.).

A variety of commercially available in vitro transcription andtranslation reagents are commercially available, including thePROTEINscript-PRO™ kit (for coupled transcription/translation) the wheatgerm IVT kit, the untreated reticulocyte lysate kit (each from Ambion,Inc (Austin Tex.)), the HeLa Nuclear Extract in vitro Transcriptionsystem, the TnT Quick coupled Transcription/translation systems (bothfrom Promega, see, e.g., Technical bulletin No. 123 and Technical ManualNo. 045), and the single tube protein system 3 from Progen. Each ofthese available systems (as well as many other available systems) havecertain advantages which are detailed by the product manufacturer.

In addition, the art provides considerable detail regarding the relativeactivities of different in vitro transcription translation systems, forexample as set forth in Tymms, id.; Jermutus et al. (1999) “Comparisonof E. Coli and rabbit reticulocyte ribosome display systems” FEBS Lett.450(1-2):105-10 and the references therein; Jermutus et al. (1998)“Recent advances in producing and selecting functional proteins by usingcell-free translation” Curr. Opin. Biotechnol. 9(5):534-48 and thereferences therein; Hanes et al. (1988) “Ribosome Display EfficientlySelects and Evolves High-Affinity Antibodies in vitro from ImmuneLibraries” PNAS 95:14130-14135 and the references therein; and Hanes andPluckthun (1997) “In vitro Selection and Evolution of FunctionalProteins by Using Ribosome Display.” Biochemistry 94:4937-4942 and thereferences therein.

For example, an untreated rabbit reticulocyte lysate is suitable forinitiation and translation assays where the prior removal of endogenousglobin mRNA is not necessary. The untreated lysate translates exogenousmRNA, but also competes with endogenous mRNA for limiting translationalmachinery.

Similarly, The PROTEINscript-PRO™ kit from Ambion is designed forcoupled in vitro transcription and translation using an E. coli S30extract. In contrast to eukaryotic systems, where the transcription andtranslation processes are separated in time and space, prokaryoticsystems are coupled, as both processes occur simultaneously. Duringtranscription, the nascent 5′-end of the mRNA becomes available forribosome binding, allowing transcription and translation to proceed atthe same time. This early binding of ribosomes to the mRNA maintainstranscript stability and promotes efficient translation. Coupledtranscription: translation using the PROTEINscript-PRO Kit is based onthis E. coli model.

The Wheat Germ IVT™ Kit from Ambion, or other similar systems, is/are aconvenient alternative, e.g., when the use of a rabbit reticulocytelysate is not appropriate for in vitro protein synthesis. The Wheat GermIVT™ Kit can be used, e.g., when the desired translation productcomigrates with globin (approx. 12-15 kDa), when translating mRNAscoding for regulatory factors (such as transcription factors or DNAbinding proteins) which may already be present at high levels inmammalian reticulocytes, but not plant extracts, or when an mRNA willnot translate for unknown reasons and a second translation system is tobe tested.

The TNT® Quick Coupled Transcription/Translation Systems (Promega) aresingle-tube, coupled transcription/translation reactions for eukaryoticin vitro translation. The TNT® Quick Coupled Transcription/TranslationSystem combines RNA Polymerase, nucleotides, salts and RecombinantRNasine Ribonuclease Inhibitor with the reticulocyte lysate to form asingle TNT® Quick Master Mix. The TNT® Quick CoupledTranscription/Translation System is available in two configurations fortranscription and translation of genes cloned downstream from either theT7 or SP6 RNA polymerase promoters. Included with the TNT® Quick Systemis a luciferase-encoding control plasmid and Luciferase Assay Reagent,which can be used in a non-radioactive assay for rapid (<30 seconds)detection of functionally active luciferase protein.

In addition to coupled in vitro transcription and translation, eitherstep may be done separately from the other by in vitro or cellularmeans. For example, in vitro transcribed RNA can be provided to cellsfor subsequent translation by way of mechanical or osmoticmicroinjection, methods for which are well known in the art. Moreover,cells containing RNA derived by transcription from one or more of theshuffling and mutagenesis methods described (directly or indirectly)herein can be lysed and the RNA obtained for subsequent analysis. Thepurified or unpurified RNA obtained in this manner can be subjected toin vitro or in situ translation. All such methods can be conductedwithin or in conjunction with the various arraying approaches describedin this invention.

Many other systems are well known, well characterized and set forth inthe references noted herein, as well as in other references known to oneof skill. It will also be appreciated that one of skill can producetranscription/translation systems similar to those which arecommercially available from available materials, e.g., as taught in thereferences noted above.

The methods of the invention can include in-line or off-linepurification of one or more reaction product array members. In linepurification is performed as part of the transfer process from an invitro transcription/translation reaction to a product detection oridentification module, whereas off-line purification can be performedbefore or after transfer, or in a parallel module.

In any case, once expressed, proteins can be purified, either partiallyor substantially to homogeneity, according to standard procedures knownto and used by those of skill in the art. Polypeptides of the inventioncan be recovered and purified from arrays by any of a number of methodswell known in the art, including ammonium sulfate or ethanolprecipitation, acid or base extraction, column chromatography, affinitycolumn chromatography, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,hydroxylapatite chromatography, lectin chromatography, gelelectrophoresis and the like. Protein refolding steps can be used, asdesired, in completing configuration of mature proteins. Highperformance liquid chromatography (HPLC) can be employed in finalpurification steps where high purity is desired. Once purified,partially or to homogeneity, as desired, the polypeptides may be used(e.g., as assay components, therapeutic reagents or as immunogens forantibody production).

In addition to the references noted supra, a variety ofpurification/protein folding methods are well known in the art,including, e.g., those set forth in R. Scopes, Protein Purification,Springer-Verlag, N.Y. (1982); Deutscher, Methods in Enzymology Vol. 182:Guide to Protein Purification, Academic Press, Inc. N.Y. (1990); Sandana(1997) Bioseparation of Proteins, Academic Press, Inc.; Bollag et al.(1996) Protein Methods, 2^(nd) Edition Wiley-Liss, NY; Walker (1996) TheProtein Protocols Handbook Humana Press, NJ, Harris and Angal (1990)Protein Purification Applications: A Practical Approach IRL Press atOxford, Oxford, England; Harris and Angal Protein Purification Methods:A Practical Approach IRL Press at Oxford, Oxford, England; Scopes (1993)Protein Purification: Principles and Practice 3^(rd) Edition SpringerVerlag, NY; Janson and Ryden (1998) Protein Purification: PrinciplesHigh Resolution Methods and Applications, Second Edition Wiley-VCH, NY;and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ; and thereferences cited therein. Additional details regarding protein foldingand other in vitro protein biosynthetic methods are found in Marszal etal. U.S. Pat. No. 6,033,868 (Mar. 7, 2000).

As noted, those of skill in the art will recognize that after synthesis,expression and/or purification, proteins can possess a conformationsubstantially different from the native conformations of the relevantparental polypeptides. For example, polypeptides produced by prokaryoticsystems often are optimized by exposure to chaotropic agents to achieveproper folding. During purification from, e.g., lysates derived from E.coli, the expressed protein is optionally denatured and then renatured.This is accomplished, e.g., by solubilizing the proteins in a chaotropicagent such as guanidine HCl.

In general, it is occasionally desirable to denature and reduceexpressed polypeptides and then to cause the polypeptides to re-foldinto the preferred conformation. For example, guanidine, guanidinium,urea, detergents, chelating agents, DTT, DTE, and/or a chaperonin can beadded incubated with a transcription product of interest. Methods ofreducing, denaturing and renaturing proteins are well known to those ofskill in the art (see, the references above, and Debinski, et al. (1993)J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug.Chem., 4: 581-585; and Buchner, et al., (1992) Anal. Biochem., 205:263-270). Debinski, et al., for example, describe the denaturation andreduction of inclusion body proteins in guanidine-DTE. The proteins canbe refolded in a redox buffer containing, e.g., oxidized glutathione andL-arginine. Refolding reagents can be flowed or otherwise moved intocontact with the one or more polypeptide or other expression product, orvice-versa:

Various systems are also available for simultaneous synthesis andfolding of complex proteins. For example, the control of redoxpotential, the use of helper proteins (from both bacterial andeukaryotic systems) and the like can be used to provide for improvedcell free translation. Optionally, proteins may be added which aid inprotein refolding, such as by maintaining solubility of the nascent orpartially folded protein (e.g., chaperonins) or by adjusting theconfiguration of inter- and intra-molecular disulfide bonds (e.g.protein disulfide isomerase).

RNA or protein or other products of a translation reaction can be taggedwith any available tag (biotin, His tag, etc.), and captured to an arrayposition following expression, if desired. The products are released,e.g., by cleavage of an incorporated cleavage site, or other releasingmethods (salt, heat, acid, base, light, or the like). In alternateembodiments, products are free in solution or encapsulated inmini-reaction compartments such as inverted micelles, liposomes, or gelparticles or droplets.

As noted, it can be desirable to reconstitute expression products inliposomes, inverted micelles, or other lipid systems. Thus, the systemcan include a source of one or more lipid. Typically this lipid isflowed into contact with the one or more polypeptide or other reactionproduct (or vice-versa), or into contact with the physical or logicalarray of reaction mixtures. Similarly, the lipid can be flowed intocontact with one or more shuffled or mutagenized nucleic acids (ortranscription products thereof), thereby producing one or more liposomesor micelles comprising the polypeptide or other reaction product,reaction mixture components, and/or nucleic acids.

Liposomes and related structures are particularly attractive systems foruse in the present invention, because they serve to concentrate reagentsof interest into small volumes and because they are amenable to FACS andother high-throughput methods. In addition to standard FACS methods,microfabricated FACSs for use in sorting cells and certain subcellularcomponents such as molecules of DNA have also been described in, e.g.,Fu, A. Y. et al. (1999) “A Microfabricated Fluorescence-Activated CellSorter,” Nat. Biotechnol. 17:1109-1111; Unger, M., et al. (1999) “SingleMolecule Fluorescence Observed with Mercury Lamp Illumination,”Biotechniques. 27:1008-1013; and Chou, H. P. et al. (1999) “AMicrofabricated Device for Sizing and Sorting DNA Molecules,” Proc.Nat'l. Acad. Sci. 96:11-13. These sorting techniques utilizingmicrofabricated FACSs generally involve focusing cells usingmicrochannel geometry and can be adapted to the present invention by theinclusion of a chip-based FACS system in the in vitrotranscription/translation module of the system.

The following example provides details regarding use of liposomes asreaction vesicles.

(1.) Alternate Format: In Vitro Clone Selection: Direct Isolation ofActive Sequences from a DNA Library—Use of Liposomes in the IntegratedSystems of the Invention

The slowest step in the manipulation of DNA is often the selection offunctional DNA constructs in vivo. That is, DNA is often maintained in aform suitable for transformation and growth in a host organism, such asE. coli, to allow the selection of positive constructs from thebackground. This example describes functional assays to be performed onthe gene product, which is transcribed directly from a DNA library,leading to the isolation of the specific construct bearing the desiredactivity. The technique is amenable to the screening of libraries of anysize.

This example relies upon the application of a number of techniques inseries. In particular, the example uses liposomes as reaction/sortingcompartments, in vitro transcription/translation, a fluorescent activityassay and a FACS machine.

The use of in vitro transcription/translation systems to produce smallamounts of protein from DNA in solution is described above. Theencapsulation of this machinery inside a small compartment (−1 μm), suchas an inverted micelle (Tawfik and Griffiths (1998) Nature Biotech,16:652-656) or liposome, enables the machinery to act upon a single DNAmolecule. The presence of 1 molecule in a 1 μm diameter spherecorresponds to a concentration of ˜2.5 nM. Thus, the effectiveconcentration of the DNA is sufficient for efficienttranscription/translation and even a single round of translation gives auseful protein concentration. A single turnover of the enzyme encoded bythe DNA also gives nM concentrations of product; therefore, e.g., about100 catalytic events are sufficient for detection. Detection of thisfluorescence by the laser of the FACS machine will then lead to thesorting of the fluorescent compartments (liposomes only, as invertedmicelles are incompatible with the FACS machine). In general, FACSmachines sort liposomes, cells or other sortable compartments at a rateof thousands per second, which allows millions of liposomal reactioncompartments to be sorted routinely. The selected liposomes can then bedegraded and the formerly encapsulated DNA isolated and purified. TheDNA that encoded a gene product(s) capable of generating fluorescenceunder the assay conditions are substantially present in this sample.This DNA is further analyzed or used directly in another cycle of thisprocess under more stringent conditions.

For example, Tawfik and Griffiths, id, describes a system in whichlinear DNA encoding a DNA methylase was isolated from a background ofother DNA. The DNA was encapsulated in inverted micelles with suitabletranscription/translation machinery, such that only one DNA molecule wasencapsulated in each micelle. After the DNA methylase had beentranslated, it methylated the DNA accessible to it, i.e. present in thatmicelle. The reaction was quenched and the DNA was isolated from themicelles. The pooled DNA was then exposed to the restriction enzymecorresponding to the methylase, leading to the degradation ofunmethylated sequences. The intact DNA was then amplified by PCR and theDNA was found to be highly enriched in the methylase encoding sequence.

A solution of the in vitro transcription/translation machinery with thesubstrates required for the activity assay is provided, atconcentrations sufficient to ensure that each liposome contains a selfsufficient transcription/translation/gene product assay system, in asuitable buffer, at 4° C. A DNA library is added at a concentration suchthat generally only about one or zero DNA molecule(s) are present ineach liposome.

The liposomes are formed using a solvent dispersal method, which allowsthe direct formation of small unilamellar vesicles of defined size inthe starting solution. The starting solution is stirred at apredetermined speed and the lipids are added to the solution in a watermiscible solvent. As the solvent disperses (solvent is typically lessthan 2% final concentration) the lipids are exposed to the aqueous phasewhich causes them to spontaneously form SUVs of a size defined by theconditions and the choice of lipid mixture. In a typical experiment 30%of the initial solution will be encapsulated in liposomes. The liposomesare purified and the remaining unencapsulated solution can be recycledif desired. The liposomes are then incubated under conditions that favortranscription/translation and later conditions suitable for the activityassay of interest.

The stability of the liposomes and their behavior in solution can becontrolled by the choice of the constituent lipids, which form thebilayer. Thus, the compartment for reaction can be tailored to fit theconditions necessary for a specific experiment. Fluorescent lipids canalso be incorporated into the bilayer, which can be used as an internalstandard for fluorescence produced in the gene product assay e.g., inthe FACS machine.

Gene product can be assayed using any of the standard fluorescentformats, such as the production/consumption of a fluorophore in thereaction, fluorescence resonance energy transfer (FRET), or coupledassays that use the product of the reaction performed by the geneproduct as the substrate for another reaction which generates afluorophore. The tiny volume of reaction (−4 femtolitres for a ˜1 μmdiameter vesicle) increases the sensitivity of the solution to changesin the number of ions such as H⁺ (i.e. pH) and Ca²⁺ for which specificfluorescent detection methods are available. Fluorescent methods are themost commonly used assays for most enzyme classes, which providesgeneral utility for this system.

Once sufficient time has been allowed for the gene product to performits reaction, the liposome suspension is sorted using a FACS machine.Particles of ˜1 μM diameter are readily visualized/sorted at a rate ofthousands per second by this technology. Thus, the liposomes which aresufficiently fluorescent (and thus contain an active gene product/DNAconstruct) are separated from the many which do not meet the predefinedcriteria. The DNA is then purified from the sorted liposome populationusing standard methodology.

This approach confers a number of advantages over traditional cloningprotocols. Firstly, the entire screening process is performed in asingle batch, limiting the amount of liquid handling steps, so thatthere is virtually no limit to the size of library that can be screenedin a single run. The only time the individual DNA constructs are handledindividually is when they are sorted in the FACS machine, allowingextremely high throughput screens to be performed. Further, any geneproduct are handled equally efficiently, with no problems associatedwith host organism toxicity, protease mediated degradation, or the like.Even membrane associated proteins are screenable, due to the lipidbilayer nature of the liposomes.

Equally powerful particle screening methods are available by use ofquantitative (e.g. digital) imaging in association with visible orfluorescent microscopy. In such methods, a library of particlesproducing a quantifiable emission are distributed on a surface in such away as to maintain a reasonably fixed positions. Visualization andquantification of emission of light from particle(s) or specifiedsub-area(s) (as in a grid) is conducted by one of a variety of availableof microscopic devices operatively linked to and digital imaging camera.Optionally, these components may be linked to a computer or otherhigh-speed computational device equipped with software capable ofcorrecting for lens curvature, unequal background within the field ofview, and the like. Such imaging hardware and software can be used toguide (manually or electronically) the selective ‘picking’ or removal ofparticles from the surface. Such particles are then processed,characterized and arrayed as described elsewhere within this disclosure.Particularly useful for the selective ‘picking’ of particles from asurface are micromanipulation tools such as capillary-actuated clampingdevices such as find use in ion channel and patch clamp studies, opticaland atomic tweezers, micropipets, syringes, and the like.

Furthermore, because the only components in the system are added bydesign, there is no interference from overlapping activities of otherproteins, etc., leading to a low background and the ability to detectvery low levels of activity. Similarly, because no living organism isinvolved in the process, sensitive or dangerous gene products such asantibiotic resistance genes and factors which mediate infection can bestudied without risk of transferring the new activity to pathogens and,therefore, the safety concerns for the systems are relatively reduced.Finally, results of an experiment can be produced quickly withoutwaiting for an incubation period, especially when the host organism is aslow growing yeast or mold.

In addition to liposomes, individual or pooled nucleic acid populationswith relevant in vitro transcription or translation reagents may beencapsulated within agar, agarose, carageenan, guar and relatedbiological gels and gums; or in a wide variety of hygroscopic syntheticpolymers such as polyacrylates, polymethylmethacrylates,polyacrylamides, polyethyleneimine (crosslinked) membranes, or the like.Methods for using these substances to encapsulate biological materialsare known in the art. For example, microdroplets are formed by flowing amixture of the polymerizing or pre-gelled polymer with a mixturecontaining the biochemical components of interest. Microdroplettechnology is described, e.g., in Weaver et al. (1993) “Microdroptechnology: A General Method for Separating Cells by Function andComposition” METHODS: A Companion to Methods in Enzymology 2(3)234-247).

The resulting mixture is passed through a mechanical or aspiratingdevice capable of atomizing the stream into microdroplets of desiredsize or characteristics. Such microdroplets can be sprayed onto asurface, plate, preformed grid, or the like, directly from the atomizingdevice, or passed into a separate aspirator, nozzle or ink jet-likedevice. Commonly, the particles can be sprayed in a random orsemi-random manner onto the target surface and allowed to retain arelatively fixed position either by surface tension, gel adhesion ormaintenance of a low moisture or low-eddy current capillary layer on agel or moist surface. The positions of the quantified particles may beused to establish and record an initial array or the particles ofinterest may be picked and repositioned in a more normal pattern toestablish the functional array.

This embodiment facilitates the process of developing biologicalcatalysts for novel functions by giving a direct connection between DNAstructure and gene product activity and by decreasing the time requiredfor the interactive evolution of novel activities.

(2.) Alternate Format: Localizing In Vitro Transcription/TranslationProducts

Methods of detecting or enriching for in vitro transcription ortranslation products are provided. In the methods, one or more firstnucleic acids (e.g., shuffled or otherwise diversified nucleic acids)which encode one or more moieties are localized proximal to one or moremoiety recognition agents which specifically bind the one or moremoieties. The one or more nucleic acids are in vitro translated ortranscribed, producing the one or more moieties (e.g., polypeptides orbiologically active RNAs such as anti-sense or ribozyme molecules, orother product molecules). The one or more moieties diffuse or flow intocontact with the one or more moiety recognition agents (e.g.,antibodies, antigens, etc.). Binding of the one or more moieties to theone or more moiety recognition agents is permitted and the one or moremoieties are detected or enriched for by detecting or collecting one ormore materials proximal to, within or contiguous with the moietyrecognition agent (the material comprises at least one of the one ormore moieties, where the moieties comprise one or more in vitrotranslation or transcription product). Optionally, the one or moremoieties are pooled by pooling the material which is collected. Hereagain, a variety of variants of this basic class of methods are setforth herein as are a variety of products produced by the methods andtheir variants. The one or more moieties can be pooled by pooling thematerial which is collected.

For example, the first nucleic acids can include a related population ofshuffled nucleic acids which encode an epitope tag, which is bound bythe moiety or one or more moiety recognition agents. The first nucleicacids can include transcription or translation control sequences, suchas an inducible or constitutive heterologous (or non-heterologous)promoter. In some embodiments, the first nucleic acids include a relatedpopulation of shuffled nucleic acids and a PCR primer binding region,the method further including PCR amplifying a set of parental nucleicacids to produce the related population of shuffled nucleic acids.

Optionally, the first nucleic acids can include a related population ofshuffled nucleic acids and a PCR primer binding region. In this case,the method can include identifying one or more target first nucleic acidby proximity to the moieties which are bound to the one or more moietyrecognition agent, and amplifying the target first nucleic acid byhybridizing a PCR primer to the PCR primer binding region and extendingthe primer with a polymerase.

The first nucleic acids and the one or more moiety recognition agentscan be localized on a solid substrate (including membranes, beads andother substrates commonly available), or in a gel or other matrix thatlimits diffusion of the moiety recognition agents or the nucleic acids.The first nucleic acids and the one or more moiety recognition agentscan be localized on the solid substrate by a cleavable linker, achemical linker, a gel, a colloid, a magnetic field, an electricalfield, a combination thereof, or the like. In one aspect, the moiety ormoiety in contact with the moiety recognition agent can release thenucleic acid, e.g., where the moiety recognition agent cleaves acleavable linker which attaches the first nucleic acid to a solidsubstrate.

Typically, the invention can include detecting an activity of the moietyor moiety recognition agent. The one or more first nucleic acid can thenbe picked with an automated robot, providing for recovery of the nucleicacid and further processing. For example, the one or more first nucleicacid can be picked by placing a capillary on a region comprising thedetected activity of the moiety or moiety recognition agent andwithdrawing the capillary.

Example: Enrichment Method for In Vitro Transcription/TranslationProducts

FIG. 17, Panels A-E schematically show an embodiment in which productsof in vitro transcription/translation (ivTT) are captured on a solidsubstrate or in a matrix for further analysis, e.g., via immobilizedantibodies or other protein capture mechanisms. As shown, both in vitrotranscription and translation products can be captured on a singlesubstrate, providing a mechanism for direct identification and isolationof genes of interest on the substrate.

As shown, an oligonucleotide “hook” is used to capture shuffled orotherwise diversified genes (the hook can hybridize to a region that isheld constant in the shuffling or other diversification reaction) to thesubstrate (which may be any of the substrates herein, including beads,membranes, slides, trays, etc.). Alternately, the oligo can bind auniversal epitope on a PCR primer of interest that is incorporated intothe gene, e.g., a biotin or other molecule. The gene is in vitrotranscribed/translated, with the product being captured by anappropriate binding moiety (if the product is a protein, an antibody canbe used as the binding moiety; if the product is an RNA, a secondcapture nucleic acid can be used as the binding moiety). For example,the surface (e.g., plate/bead/well) can be coated with oligos,antibodies, or both. For oligo capture tags, the sequences optionallybind to generic sequence handles. The tags can include a variety offeatures, including primer sequences for PCR. The oligos can includefeatures for direct capture such as biotin or any other tag that can belinked to the oligo, e.g., through a chemical linkage, which optionallycan include a linker region. The oligos can be cleavable (e.g., throughincubation with a restriction enzyme). Similarly, cleavage itself can bea marker of activity, e.g., where activity of a restriction enzyme orvariant is the molecule to be tested. Similarly, the activity to betested can be a reporter system that results in cleavage of the capturetag. In the case of antibody tags, the tags can provide for uniformdisplay of active sites and can be used in a project independentfashion, e.g., in any system where the antibody ligand is present.

As shown, the product binds to the binding moiety in proximity to thecaptured gene. Any activity of the product is then detected. The codingnucleic acid is isolated by its proximity to the detected product, e.g.,using a microcapillary or the like. For example, the product can producea visible signal when active and the system can detect the signal (e.g.,by signal region size, signal intensity, etc.) and select thecorresponding region for isolation of the coding nucleic acid. Inbead-based embodiments, nucleic acids can be selected by FACS or otherfluorescence detection methods. The use of the hook to capture DNAoffers many control point options, including, e.g., cleavage by avariant.

In one embodiment, which is shown in FIG. 17 B, the product has anactivity which results in cleavage of proximal bound coding nucleicacids. However, depending on the nature of the substrate or matrix, anyavailable method can be used for cleavage of the coding nucleic acid,including chemical cleavage, light-directed cleavage, treatment with arestriction enzyme, or the like. The oligonucleotide hook can alsoinclude a cleavable linking element, as is common in the art.

As shown, genes are transcribed from a promoter such as a T7 promoter,translated and the activity of the encoded variant enzyme detected. Inthe format depicted, the variant enzyme includes a capture region thatpermits immobilization and detection. Free (e.g., soluble) genestranscribed in the same region are isolated. The process is repeateduntil a desired enrichment is observed. The tether on the gene or thetranscribed enzyme or the constant region of the enzyme variant can becleaved, e.g., specifically. Such specifically cleaved materials can bespecifically eluted or otherwise isolated from the system. Examples ofsuch cleavable linkers include a cleavable substrate or substrateanalog, e.g., for detection of an activity of the variant protein (e.g.,upon binding/cleavage by the protein variant, e.g., where the protein isan enzyme). Similarly, cleavage can be dependent on formation of adesired side product such as peroxide, heat, light, electricity or thelike.

It is helpful to limit diffusion in this system, because, as thetranscription and/or translation product diffuses away from the tetheredcoding gene, the association between the tethered gene and the encodedproducts becomes more difficult to determine. Diffusion can be limitedby any available method, including allowing fortranscription/translation in a matrix that limits diffusion (e.g., a gelor polymer solution).

FIG. 17, panel C shows details of one embodiment using generic epitopetags. As shown, the tags provide for uniform display of the variousactive sites of the protein or other bio-molecule of interest. Thisprovides for project independent use of the tags as well as for the useof common reagents. Common tags such as His-tag IMAC can be used, as canany fusion protein comprising a region to be used as tags. The systemalso provides for common treatment such as free thiol introduction andthe like.

As shown in FIG. 17, panel D, a robotic system such as the commerciallyavailable Q-bot can be used to pick positive regions of the substrate(e.g., to capture free genes prior to diffusion from a site of interest.Picking can be performed according to any standard hit picking selectioncriteria, e.g., selection of a particular percentage of variants by thesize/intensity produced by a product at a site of activity/expression.Alternately, a bead based protocol can be used in conjunction with FACSif a fluorescent product is formed. In either case, genes which areselected can be used as inputs for subsequent rounds of recombination ormutation (or both) and screening, or can simply be used as productcandidates. The products can also be further screened, in pools or assingle hits, using any appropriate assay.

As shown in FIG. 17, panel E, DNAs which are recovered are subject toamplification reactions such as PCR or LCR and the amplified productssubject to any additional diversity generation, isolation or selectionstep which is selected by the user or the system. As depicted, recoveryin this example is performed via a microcapillary approach (e.g., usingthe Q-bot) and then subject to RT-PCR to produce products that, againcan be used in subsequent recombination/mutation procedures or for anyof the other purposes noted herein. It is worth noting that the densityof variant genes of interest is inversely proportional to the enrichmentof components in the system. Thus, to avoid bystander effects, thedensity of variant genes should not be too high for accurate selectionby whatever selection mechanisms are used (capillary, FACS, etc.).

These methods can also be adapted to in vivo systems by lysing cells andcapturing cell components. Systems for cell lysis and capture of nucleicacids such as Xpress-Screen™ from Tropix PE Biosystems (Bedford Mass.)can be adapted for use with this embodiment of the invention.

C. High-Throughput Cloning and Expression

In addition to in vitro transcription/translation, high throughputcloning and expression can be used to generate products to screen forproduct activity. This approach has the advantage of expressing productsin a system that is similar to the eventual intended expression site formany products (e.g., in cells).

Basic cloning methodology is set forth in Sambrook, Ausubel and Berger,supra. In the present high-throughput system, diversified nucleic acids(e.g., a shuffled DNAs) are transformed into cells. The cells are sorted(e.g., by FACS, micro-FACS, visual or fluorescence microscopy) byexpression of a marker protein such as GFP, where the marker expressionis encoded by a full-length copy of a corresponding nucleic acid, e.g.,where the full-length nucleic acid also encodes a full-length product ofinterest. Cells that have been selected are transferred to amicro-chamber or array where they express the shuffled gene. Themicro-chamber or array contains a substrate for the shuffled proteinwhose optical properties (i.e. absorbance or fluorescence) are changedby catalysis by the enzyme. After a period of time, (e.g., ca. minutesto hours) the array of micro-chambers is “read” with a laser, CCD cameraor other high density optical device. Those chambers in which the changein optical properties exceeds some threshold (i.e. a defining activity)are emptied, one into each well of a high density microtitre plate (96,384, 1500 well etc), and the cells are then grown for the second assay.This provides a high-throughput format as a pre-screen for activeclones.

Cells containing shuffled or mutated genes can express a protein orpathway capable of providing a florescent signal directly. In such acase, the cell supplies the translation and, optionally, thetranscriptional machinery, and required substrates are loaded byincubating cells in a mixture appropriate for delivering the substratethrough the cell wall. Cells expressing either marker or library genesof interest are sorted and arrayed or collected on the basis of theemitted fluorescence signal. Such a signal may also derive from thescattering, or direct emission or absorbance of visible light from theindividual cells.

Several alternatives to traditional FACS devices exist and provideparticularly unique advantages to the present invention. For example,microfluidic systems (see, e.g., Fu A Y, Spence C, Scherer A, Arnold F Hand Quake S R., (1999) “A microfabricated fluorescence-activated cellsorter” Nat. Biotechnol. 17(11):1109-11) provide an efficientalternative to traditional FACS devices. Such systems are typicallymicrofabricated devices capable of flowing, detecting and sorting cellsfrom a microfluidic stream. Such systems can have several advantagesover traditional FACS in that they allow for reversible fluid flow,extraordinarily high sorting accuracy, parallel sorting of multiplesamples and the sorting of particles which are below the limit ofconventional FACS devices. (e.g. bacteria, phage, phagemids,sub-microparticles, and the like).

In addition, a variety of powerful particle and cell screening methodsare available by use of quantitative (e.g. digital) imaging inassociation with visible or fluorescent microscopy. In such methods, alibrary of cells producing quantifiable emission(s) are distributed on asurface in such a way as to maintain a reasonably fixed positions.Visualization and quantification of emission of light from each particleor specified sub-area (as in a grid) is conducted by one of a variety ofavailable of microscopic devices operatively linked to and digitalimaging camera. Optionally, these components may be linked to a computeror other high-speed computational device equipped with software capableof correcting for lens curvature, unequal background within the field ofview, and the like. Such imaging hardware and software can be used toguide (manually or electronically) the selective ‘picking’ or removal ofparticles from the surface. Such particles are then processed,characterized and arrayed as described elsewhere within this disclosure.Particularly useful for the selective ‘picking’ of particles from asurface are micro-manipulation tools such as capillary-actuated orsuction-actuated clamping devices, such as find use in ion channel andpatch clamp studies, optical and atomic tweezers, micropipets andsyringes, and the like.

D. Product Deconvolution

During operation of the device, the array of reaction mixtures producesan array of reaction mixture products (e.g., biologically active nucleicacids or proteins). These biologically active nucleic acids or proteinsare screened for at least one property to identify coding nucleic acidsof interest. Thus, in one significant aspect, the device or integratedsystem herein has one or more product identification or purificationmodules.

These product identification/purification modules identify and/or purifyone or more members of the array of reaction mixture products.

Common methods of assaying for product activity include any of thoseavailable in the art, including enzyme and/or substrate assays,cell-based assays, reporter gene expression, second messenger inductionor signaling, etc.

In addition to product identification or purification, productidentification or purification modules can also include an instructionset for discriminating between members of the array of reaction productsbased upon detectable characteristics, such as a physical characteristicof the products, an activity of the products or reactants, andconcentrations of the products or reactants. For example “hit picking”software is available which permits the user to select criteria toidentify members of an array that display one or more activity which issufficient to be of interest for further analysis.

The product identification module can include detection and/or selectionmodules which facilitate detection or selection of array members. Suchmodules can include, e.g., an array reader which detects one or moremember of the array of reaction products. Array readers are commerciallyavailable, generally constituting a microscope or CCD and a computerwith appropriate software for identifying or recording information. Inparticular, array readers which are designed to interface with standardmicrotiter trays and other common array systems are commerciallyavailable. In addition to product manufacturer information from many ofthe various product manufacturers noted herein, detection protocols andsystems are well known. For example, basic bioluminescence methods anddetection methods which describe e.g., detection methods include LaRossaEd. (1998) Biolumniescence Methods and Protocols: Methods in MolecularBiology Vol. 102, Humana Press, Towata, N.J. Basic Light microscopymethods, including digital image processing is described, e.g., inShotton (ed) (1993) Electronic Light Microscopy: Techniques in ModernBiomedical Microscopy Wiley-Liss, Inc. New York, N.Y. FluorescenceMicroscopy methods are described, e.g., in Hergman (1998) FluorescenceMicroscopy Bios Scientific Publishers, Oxford, England. Specializedimaging instruments and methods for screening large numbers of imageshave also been described, e.g., “MICROCOLONY IMAGER INSTRUMENT FORSCREENING CELLS EXPRESSING MUTAGENIZED ENZYMES” U.S. Pat. No. 5,914,245to Bylina et al.; “ABSORBTION SPECTRA DETERMINATION METHOD FOR HIGHRESOLUTION IMAGING MICROSCOPE . . . ” U.S. Pat. No. 5,859,700 to Yang;“CALIBRATION OF FLUORESCENCE RESONANCE ENERGY IN MICROSCOPY . . . ” WO9855026 (Bylina et al.); “OPTICAL INSTRUMENT HAVING A VARIABLE OPTICALFILTER” Yang and Youvan U.S. Pat. No. 5,852,498; Youvan (1999) “ImagingSpectroscopy and Solid Phase Screening” IBC World Congress on EnzymeTechnologies. These systems can be incorporated into the presentinvention to provide high-throughput screening systems.

Similarly, such modules can include any of: an enzyme which converts oneor more member of the array of reaction products into one or moredetectable products; a substrate which is converted by the one or moremember of the array of reaction products into one or more detectableproducts; a cell which produces a detectable signal upon incubation withthe one or more member of the array of reaction products; a reportergene which is induced by one or more member of the array of reactionproducts; a promoter which is induced by one or more member of the arrayof reaction products, which promoter directs expression of one or moredetectable products; an enzyme or receptor cascade which is induced bythe one or more member of the array of reaction products or the like.

Further, where a non-standard array format is used, or were non-standardassays are to be detected by the array reader, common detector elementscan be used to form an appropriate array reader. For example, commondetectors include, e.g., spectrophotometers, fluorescent detectors,microscopes (e.g., for fluorescent microscopy), CCD arrays,scintillation counting devices, pH detectors, calorimetry detectors,photodiodes, cameras, film, and the like, as well as combinationsthereof. Examples of suitable detectors are widely available from avariety of commercial sources known to persons of skill.

Signals are preferably monitored by the array reader, e.g., using anoptical detection system. For example, fluorescence based signals aretypically monitored using, e.g., in laser activated fluorescencedetection systems which employ a laser light source at an appropriatewavelength for activating the fluorescent indicator within the system.Fluorescence is then detected using an appropriate detector element,e.g., a photomultiplier tube (PMT), CCD, microscope, or the like.Similarly, for screens employing colorometric signals,spectrophotometric detection systems are employed which detect a lightsource at the sample and provide a measurement of absorbance ortransmissivity of the sample. See also, The Photonics Design andApplications Handbook, books 1, 2, 3 and 4, published annually by LaurinPublishing Co., Berkshire Common, P.O. Box 1146, Pittsfield, Mass. forcommon sources for optical components.

In alternative aspects, the array reader comprises non-optical detectorsor sensors for detecting a particular characteristic of the system. Suchsensors optionally include temperature sensors (useful, e.g., when aproduct produces or absorbs heat in a reaction, or when the reactioninvolves cycles of heat as in PCR or LCR), conductivity, potentiometric(pH, ions), amperometric (for compounds that can be oxidized or reduced,e.g., O₂, H₂O₂, I₂, oxidizable/reducible organic compounds, and thelike), mass (mass spectrometry), plasmon resonance (SPR/BIACORE),chromatography detectors (e.g., GC) and the like.

For example, pH indicators which indicate pH effects of receptor-ligandbinding can be incorporated into the array reader, where slight pHchanges resulting from binding can be detected. See also, Weaver, etal., Bio/Technology (1988) 6:1084-1089.

As noted, one conventional system carries light from a specimen field toa CCD camera. A CCD camera includes an array of picture elements(pixels). The light from the specimen is imaged on the CCD. Particularpixels corresponding to regions of the substrate are sampled to obtainlight intensity readings for each position. Multiple positions areprocessed in parallel and the time required for inquiring as to theintensity of light from each position is reduced. Many other suitabledetection systems are known to one of skill.

Data obtained (and, optionally, recorded) by the detection device istypically processed, e.g., by digitizing image data and storing andanalyzing the image in a computer system. A variety of commerciallyavailable peripheral equipment and software is available for digitizing,storing and analyzing a signal or image. A computer is commonly used totransform signals from the detection device into sequence information,reaction rates, or the like. Software for determining reaction rates ormonitoring formation of products, are available or can easily beconstructed by one of skill using a standard programming language suchas Visualbasic, Fortran, Basic, Java, or the like, or can even beprogrammed into simple end-user applications such as excel or Access.Any controller or computer optionally includes a monitor which is oftena cathode ray tube (“CRT”) display, a flat panel display (e.g., activematrix liquid crystal display, liquid crystal display), or others.Computer circuitry is often placed in a box which includes numerousintegrated circuit chips, such as a microprocessor, memory, interfacecircuits, and others. The box also optionally includes a hard diskdrive, a floppy disk drive, a high capacity removable drive, and otherelements. Inputting devices such as a keyboard, mouse or touch screenoptionally provide for input from a user.

In addition to array readers, the product deconvolution module caninclude enzymes which convert one or more member of the array ofreaction products into one or more detectable products, or substrateswhich are converted by the array of reaction products into one or moredetectable products, or other features that provide for detection ofproduct activity by direct or indirect detection formats. For example,the module can include cells which produce a detectable signal uponincubation with members of the array of reaction products, and reportergenes which are induced by one or more member of the array of reactionproducts. Similarly, the module can include promoters which are inducedby one or more array member and, e.g., which direct expression of one ormore detectable products. Enzyme or receptor cascades can be triggeredwhich are induced by the one or more member of the array of reactionproducts, with any of the products of the cascade serving as adetectable event.

Any available system for detecting proteins or nucleic acids or otherexpression products (directly or indirectly) can be incorporated intothe module. Common product identification or purification elementsinclude size/charge-based electrophoretic separation units such as gelsand capillary-based polymeric solutions, as well as affinity matrices,liposomes, microemulsions, microdroplets, plasmon resonance detectors(e.g., BIACOREs), GC detectors, epifluorescence detectors, fluorescencedetectors, fluorescent arrays, CCDs, optical sensors (e.g., anultraviolet or visible light sensor), FACS detectors, temperaturesensors, mass spectrometers, stereo-specific product detectors, coupledH₂O₂ detection systems, enzymes, enzyme substrates, Elisa reagents orother antibody-mediated detection components (e.g., an antibody or anantigen), mass spectroscopy, or the like. The particular system to beused depends on the system at issue, the throughput desired andavailable equipment.

In selected embodiments, the product identification or purificationmodules include one or more of: a gel, a polymeric solution, a liposome,a microemulsion, a microdroplet, an affinity matrix, a plasmon resonancedetector, a BIACORE, a GC detector, an ultraviolet or visible lightsensor, an epifluorescence detector, a fluorescence detector, afluorescent array, a CCD, a digital imager, a scanner, a confocalimaging device, an optical sensor, a FACS detector, a micro-FACS unit, atemperature sensor, a mass spectrometer, a stereo-specific productdetector, an Elisa reagent, an enzyme, an enzyme substrate an antibody,an antigen, mass spectroscopy, a refractive index detector, apolarimeter, a pH detector, a pH-stat device, an ion selective sensor, acalorimeter, a film, a radiation sensor, a Geiger counter, ascintillation counter, a particle counter, or an H₂O₂ detection system.

The product detection module can also include a substrate additionmodule which adds one or more substrate to a plurality of members of theproduct array or the secondary product array, e.g., where the producthas an activity on the substrate. In this embodiment, the device willinclude a substrate conversion detector which monitors formation of asecondary product produced by contact between the substrate and one ormore products. Formation of the product can be monitored directly orindirectly, or formation can be monitored by monitoring the substratedirectly or indirectly (e.g., formation of the product can be monitoredby monitoring loss of the substrate over time). Primary or secondaryproduct formation can be monitored chemo-, regio- or stereoselectively,or non-selectively.

Formation of the secondary product can be monitored by detectingformation of peroxide, heat, entropy, changes in mass, charge,fluorescence, luminescence, epifluorescence, absorbance or any of theother techniques previously noted in the context of primary product orproduct activity detection which result from contact between thesubstrate and the product.

Commonly, the product detector will be a protein detector and thepurification module will include protein purification means such asthose noted for product purification generally. However, nucleic acidscan also be products of the array, and can be similarly detected.

Array members can be moved into proximity to the product identificationmodule, or vice versa. For example, the product identification modulecan perform an xyz translation of either the identification module orthe array (e.g., by conventional robotics as set forth herein), therebymoving the product identification module proximal to the array ofreaction products. Similarly, the one or more reaction product arraymembers can be flowed into proximity to the product identificationmodule. In-line or off-line purification systems can purify the one ormore reaction product array members from associated materials.

Commonly detected products include one or more polypeptide orpolypeptide activity, one or more nucleic acid, one or more catalyticRNA, or one or more biologically active RNA or other nucleic acid(ribozyme, aptamer, anti-sense RNA, etc.).

As noted supra, the present invention provides for array duplication.For example, secondary product arrays can be produced by re-arrayingmembers of the reaction product array at a selected concentration ofproduct members in the secondary product array. The selectedconcentration can be approximately the same for a plurality of productmembers in the secondary product array (sometimes all of the arraymembers are plated at the same concentration, but it is also possible toplate members at different concentrations to provide multi-concentrationdatapoints, e.g., for kinetic analysis). This normalization ofconcentration simplifies analysis by the product detection module.

Further details on array copy systems, including copying of productarrays are found supra.

In addition to (or in place of) actually re-arraying materials, thedetection module (or a separate module) can include an instruction setfor determining a correction factor which accounts for variation inproduct concentration at different positions in the relevant array. Forexample, where product concentrations are known, a concentrationdependent correction can be applied to correct observed activity data.

Example: High Throughput Quantitation of Ligand Concentrations UsingSurface Plasmon Resonance

Selective molecular breeding utilizes the ability to measure thebiological activities of libraries of shuffled gene products.Quantitative or semi-quantitative high throughput (HTP) screening isused to rank clones with respect to biological activity during eachround of shuffling. Automation of this process is useful for decreasingthe cost and increasing the speed with which one could do cycles ofshuffling and screening.

A common problem with quantitation of libraries of shuffled proteins isthat the proteins are expressed at relatively low levels (typically 1 ngto 1 microgram per ml) and in crude mixtures such as bacterial extracts,mammalian transfection supernatants, in vitro translation reactions,etc. The potentially small amounts of the expressed protein relative tothe other components in the expression system can make quantitationchallenging.

Surface plasmon resonance (SPR) is an established technique formeasuring receptor-ligand interaction kinetics. See, e.g., Nieba et al.(1997) “BIACORE analysis of histadine-tagged proteins using a chelatingNTA sensor chip” Anal. Biochem. 22(2):217-218; Muller et al. (1998)“Tandem Immobilized Metal Ion Affinity Chromatography/Immunoaffinitypurification of His-tagged proteins—evaluation of two anti-His-tagmonoclonal antibodies” Anal Biochem. 259(1):54-61; Linder et al. (1997)“Specific Detection of His-tagged Proteins with Recombinant anti-His tagscFv-Phosphatase or scFv-phage fusions” Biotechniques 22(1):140-149. SPRallows one to measure these kinetics in the presence of complex mixturessuch as are present in expression supernatants. If all proteins in agiven library are tagged with an “equivalent” epitope tag and if astandard curve is established with an SPR probe, then one can derive theconcentration of an unknown tagged protein in a complex supernatant byobserving the kinetics of association with an immobilized antibody tothe tag.

Surface plasmon resonance (SPR) has been widely exploited to measure thekinetics of a soluble ligand with a cognate receptor immobilized on asurface that is suitable for SPR analysis. This technique is verysensitive (one can easily measure ligands at nanomolar concentrations)and can be performed in the presence of complex mixtures such as aretypically present in recombinant protein expression supernatants. Thetechnique measures the kinetics of association and dissociation of theligand:receptor pair. Given a standard curve, one can use kineticmeasurements or equilibrium binding values to estimate absoluteconcentrations of unknown protein samples which have a constant ligand,such as an epitope tag, that can interact with a receptor immobilized onthe sensor.

Preferably, SPR instruments are interfaced with robotic liquid handlingapparatus and the detectors are multiplexed so that they can be used in96-well formats. Although this example focuses on parallel 96- (orother) well SPR formats, a variant approach is to have one (or a few)SPR probe that are sequentially dipped into wells to serially measureprotein concentrations in each well. This can be achieved by moving theprobe from well to well (with a regeneration step in between) or bymoving the plate on a movable stage so that wells are sequentiallydelivered to the probe.

This example, schematized in FIG. 18, provides for the construction of amicrotiter tray compatible SPR device. SPR probe 18-1 is connected byfiber optic cables 18-2 to amplifier/detector 18-3. A 96 (or other)-wellarray (18-4) of SPR probes is fabricated with an anti-epitope tag (anepitope is attached to proteins in the library) antibody conjugated tothe surfaces of each of the SPR probes. The probe array is dipped into aplate containing, e.g., 96 unknown epitope tagged proteins (for a 96well format) at unknown concentrations. Incident light is beamed from asource, down fiber optic cables to probes. The reflected light is thenpiped from the probe back to the amplifier where it is quantitated. Thefraction of incident light that is reflected is sensitive to therefractive index difference between the probe and the material at theinterface between the probe and the unknown solution. Specific bindingof protein to the epitope tag increases the local index of refractionand this can be read out as a perturbation in the amount of incidentlight that is reflected. The probes can be standardized (shown as 1μg/ml, 10 μg/ml and 100 μg/ml curves) against solutions containing knownconcentrations of epitope tagged proteins. The standardized probes arethen dipped into the microtiter plate of unknown expression systemcomponents. The kinetics of association of the expressed proteins withthe antibody on the SPR probe are measured and the concentrations oftagged protein in the unknown samples is calculated by comparison withthe standard curve.

In addition to SPR, other approaches to protein detection can also beused. For example, the in vitro translated protein of interest can be afusion protein comprising a fluorescent or luminescent moiety such as aGFP protein. The amount of translated protein is proportional to thelevel of, e.g., GFP fluorescence and can be read by optical orspectroscopic methods.

Similarly, an epitope tag can be added as an invariant portion of anylibrary (e.g., any shuffled library). A fluorescently labeled antibodyto the tag is added to the translation mix and allowed to bind. Eitherthis binding changes fluorescence, e.g., by FRET quenching/dequenchingor an on line separation of antibody and protein is achieved by parallelcapillary electrophoresis (e.g., in a microfluidic chip format).

In one embodiment, a specific invariant amino acid sequence is added tothe library of shuffled proteins that encode an alpha helix whichcontains 4 Cysteine residues in a tetrahedral array. FlAsH is added tothe solution and binds to the epitope with a corresponding increase influorescence. There is no fluorescence background and so no separationis required. See also, Tsien et al (1999) “Target Protein Sequences forBinding of Synthetic Biarsenical Molecules” WO 9921013 A1.

E. Array Correspondence/Secondary Diversification Module

The system optionally includes an array correspondence module whichidentifies, determines or records the location of an identified productin the array of reaction mixture products which is identified by the oneor more product identification modules. The array correspondence modulecan also determine or record the location of at least a first nucleicacid member of an array, or a duplicate thereof, or of an amplifiedduplicate array, where the member corresponds to the location of one ormore member of the array of reaction products. Most commonly, thiscorrespondence module takes the form of a digital system having a queryfunction, and, e.g., a look-up table that records the correspondenceinformation across two or more arrays. For example, the query functioncan act on a user input to determine correspondence of array members inthe look-up table, or the system can be configured automatically toassess correspondence of any array member which meets a selectedcriteria (e.g., activity determined by the product detection module).Such correspondence modules can easily be programmed using availabledatabase or spreadsheet programs such as Microsoft Access™, MicrosoftExcel™, Paradox™, Quattro Pro™, or any other availablespreadsheet/database program.

This correspondence system can include a one or more secondary selectionmodule which selects at least one array member as a substrate for afurther diversification reaction (e.g., by shuffling). The selection isbased upon the location of a product identified by the productidentification modules and the corresponding location of thecorresponding nucleic acid array member identified by the arraycorrespondence member.

In shuffling embodiments, the secondary selection system optionallyincludes a secondary recombination element which physically contactsmembers of the starting arrays of nucleic acids, or duplicates oramplicons thereof, to each other or to additional sources of nucleicacids, thereby permitting physical recombination between the first andadditional members. In other aspects, all or part of the recombinationis performed in silico, and no physical contact is required forrecombination (or other diversity generating reactions).

a) Laboratory Information Management System

In general, data tracking can provide maintenance of the associationsbetween array elements and results which correlate to the arrayelements. For example, sets of results on projects can includeassociation of three relationships:

1. Array member ID—Data Sample ID;

2. Data Sample ID—Data Values;

3. Data Values—Processed Results.

Relationship 1 includes the association of array member names with theidentifiers of tested samples (e.g., “Plate 1, well A-4”). Relationship2 includes the association of device data output with the testedsamples. Relationship 3 includes the association of device output valueswith results.

In order to utilize systems and devices herein, an integrated sampletracking process can be used based on commercially available LIMS(Laboratory Information Management System) products. As each sample goesthrough many different formats (pooling, deconvolution, dilution, hitpicking, assorted assay formats, etc.) it is useful to have a veryflexible LIMS to capture that distribution of formats of parentalsamples and subsequent progeny samples. The generated data for eachsample is subsequently integrated with each format and accessible forthe user in conjunction with the samples' “pedigree.” The data isdisplayed through any one of many commercially available data analysissoftware such as SpotFire or ActivityBase to allow monitoring of theprocess.

For all data-generating devices, the output data can be associated withthe sample ID. In other words, each data point can be associated withthe well analyzed. This is relatively simple for most systems designedto scan microplates, such as plate readers, but can be more complex forsystems where the analytes are sampled from their container, such as inmass spectrometry and HPLC. Where necessary, custom software is used tolink data output to sample ID and output the resulting table to thedatabase in a standard format.

HTP screening generates huge amounts of data, which is preferably storedin an organized way. Where the amount of data is too large for easystorage on data servers, a system for data archival and retrieval isalso incorporated. The system can include, e.g., a table that tracksdatafiles (which can be, e.g., data folders), based on, e.g., name andID. The table has a column to store both a current location (such as ahard disk), e.g., in URL format, and a location on a backup disk. Backupdisks (CD/DVD) themselves have an ID which can be tracked. Archiving canbe done automatically, e.g., based on acquisition date or by usertriggering. Backed up files are retained on the server and flagged. Oncea backup takes place, the user can delete the file from the server.

F. Elements for Arraying and Handling Fluids in the Device

There are a number of common elements to the integrated systems hereinwhich form a “backbone” for the device. For example, the device includesarray elements, liquid handling elements, robotics (e.g., for movingmicrotiter plates) and the like.

(1.) Liquid Handler

The reactant arrays of the invention can be either physical or logicalin nature. For the generation of common arrangements involving fluidtransfer to or from microtiter plates, a fluid handling station is used.Several “off the shelf” fluid handling stations for performing suchtransfers are commercially available, including e.g., the Zymate systemsfrom Zymark Corporation (Zymark Center, Hopkinton, Mass.) and otherstations which utilize automatic pipettors, e.g., in conjunction withthe robotics for plate movement (e.g., the ORCA® robot, which is used ina variety of laboratory systems available, e.g., from Beckman Coulter,Inc. (Fullerton, Calif.).

In an alternate embodiment, fluid handling is performed in microchips,e.g., involving transfer of materials from microwell plates or otherwells through microchannels on the chips to destination sites(microchannel regions, wells, chambers or the like). Commerciallyavailable microfluidic systems include those fromHewlett-Packard/Agilent Technologies (e.g., the HP2100 bioanalyzer) andthe Caliper High Throughput Screening System. The Caliper HighThroughput Screening System provides an interface between standardlibrary formats and chip technologies. Furthermore, the patent andtechnical literature includes examples of microfluidic systems which caninterface directly with microwell plates for fluid handling.

Thus, generally, microfluidic systems are commercially available. Inaddition, university groups such as Mark Burns' research group at TheUniversity of Michigan also describe various microfluidic systems.Accordingly, general fabrication principles and the use of variousmicrofluidic systems is known and can be applied to the integratedsystems of the present invention.

(2.) Array Configurations

Any of a variety of array configurations can be used in the systemsherein. One common array format for use in the modules herein is amicrotiter plate array, in which the array is embodied in the wells of amicrotiter tray. Such trays are commercially available and can beordered in a variety of well sizes and numbers of wells per tray, aswell as with any of a variety of functionalized surfaces for binding ofassay or array components. Common trays include the ubiquitous 96 wellplate, with 384 and 1536 well plates also in common use.

In addition to liquid phase arrays, components can be stored in solidphase arrays. These arrays fix materials in a spatially accessiblepattern (e.g., a grid of rows and columns) onto a solid substrate suchas a membrane (e.g., nylon or nitrocellulose), a polymer or ceramicsurface, a glass or modified silica surface, a metal surface, or thelike. Components can be accessed, e.g., by local rehydration (e.g.,using a pipette or other fluid handling element) and fluidic transfer,or by scraping the array or cutting out sites of interest on the array.

While arrays are most often thought of as physical elements with aspecified spatial-physical relationship, the present invention can alsomake use of “logical” arrays, which do not have a straightforwardspatial organization. For example, a computer system can be used totrack the location of one or several components of interest which arelocated in or on physically disparate components. The computer systemcreates a logical array by providing a “look-up” table of the physicallocation of array members. Thus, even components in motion can be partof a logical array, as long as the members of the array can be specifiedand located.

G. DNA Shuffling on Solid Supports

For clarity, much of the preceding discussion describes the use ofliquid phase arrays such as those utilizing microtiter tray formats.However, as noted throughout, solid phase arrays represent analternative and also preferred format for performing many operations ofthe systems herein. The following is a description of exemplarysolid-phase shuffling formats.

As noted, DNA shuffling is a very powerful technique to generate diversegene libraries from known gene family members through a combination ofrecombination, mutagenesis and selection. Current DNA shuffling methodscan use primeness PCR assembly, where fragments of genes reassemblebased upon the kinetics of oligo re-annealing, which are then extendedby DNA polymerase in the presence of dNTPs.

A modification of this DNA shuffling process is performed where oligoannealing and extension by DNA polymerase proceed while theoligonucleotide, or alternatively, the single-stranded templatepolynucleotide is tethered to a solid support (or substrate). The methodbelow offers advantages to traditional solution based assembly in thatassembly occurs sequentially. Therefore, the specific fragments added ateach step can be more tightly controlled than solution based assembly.Also, this embodiment optionally combines the assembly and rescue steps,reducing the complexity of the overall shuffling process. This newapproach provides novel shuffling methods that utilize technologysimilar to the combinatorial synthesis of peptides and small molecules.

For example, one may create shuffled libraries by starting assemblyusing an oligonucleotide(s) that is/are tethered to a solid support. Theprocess typically involves tethering the oligonucleotide(s) to a solidsupport so that at least about 10-20 nucleotides including the 3′hydroxyl are exposed to solvent. In some embodiments, a synthesizermodule is used to synthesize one or more nucleic acid fragment on asolid support. Such fragments are optionally created from one or moreparental nucleic acids sequences by a computer operably coupled to thesynthesizer module.

In any case, the oligo(s) are then typically annealed to mixtures ofsingle stranded nucleic acid generated, e.g., by the processes discussedherein, for example, partial DNAse digestion of either PCR products ofseveral related genes or genomic or cDNA from homologues of interest.The annealed hybrids are extended, typically with DNA polymerase (forexample, with a thermostable DNA polymerase such as Taq DNA polymerase),generating a bound library of extended, solid-support tethered doublestranded duplexes. The bound library is denatured to release the secondstrand. The tethered oligo is reannealed to the released library ofDNAse treated fragments and extended. This process is repeated untilfragments of desired length are formed. The library of shuffled productsis released from the solid support and used as desired, e.g., for invitro transcription translation or cloning into vectors.

At any of these steps, the solid support allows one to purify thereaction products taking advantage of the properties of the solidsupport (for example, the solid support can include magnetic beads thatcan be manipulated by applying a magnetic field.

One feature of this approach is that by using an oligonucleotide ofprecise length to tether to the support (for example a 38 nt oligo) onehas pre-determined the location of the first chimera (in the example, itwill begin at nucleotide 39). This is true for the firstoligonucleotide. This feature can be useful in keeping parts of thenucleic acid constant, e.g., for cloning purposes or where a feature isnot desired to be diversified.

One can use this feature in (at least) two ways. First, if the genes arecloned into a similar vector, the first oligo can anneal to vectorsequence (for example immediately adjacent to the gene coding region).In this way, the entirety of new gene combinations are synthesized fromDNA fragments with randomly generated ends (e.g., from DNAse treatment),but the vector sequence is kept constant for cloning purposes.

Where one desires to eliminate this feature (where all nucleotides areto be varied for diversity generation purposes), one can tether amixture of oligonucleotides of varying length to the support (forexample, oligos from 35-50 nucleotides give chimeras starting in rangeof nt36 to nt51), or one can vary the sequences of the tetheredoligonucleotides to vary this region, e.g., according to the various insilico and oligonucleotide-mediated methods discussed above.

In typical DNA shuffling, extension of DNAse fragments occurs at anyplace annealing occurs. In contrast, tethering the oligo to solidsupports likely restricts the choice of oligo to those at the ends ofthe DNA of interest (although one can tether using oligos designed toregions internal to the gene of interest, ultimately the entire DNA ofinterest is usually, though not always, re-assembled, e.g., to generatea full length, or substantially full length, heterolog).

The addition of DNA fragments to the tethered oligonucleotide istypically sequential. The assembly process can be paused at any step andconditions changed. For example, one can add or subtract gene fragmentsduring the assembly. For example, one can start the assembly with genes1, 2, and 3, but remove gene 1 after initial round. Similarly,particular blends of genes can be selected at any stage to biasrecombination (at any stage) towards one or more parental type. Forexample, one can change from genes 1-4 to only genes 1 and 4 after 5extensions; or alter the representation of any gene in the recombinationprocess, e.g., change gene 1, e.g., from 1:4 to 1:2 for the last 3extensions to bias the recombination, e.g., to achieve selectable geneblending. Alternatively, one can alter PCR conditions for parts of theassembly, e.g., longer extensions at the 3′ end. This provides animproved level of control over the progress and outcome of shufflingexperiments. For example, one can add DNAse fragments corresponding tothe 5′ end of genes separately from fragments corresponding to the 3′end.

An additional feature of the invention is that assembly and rescue canoccur simultaneously. Also, the sequential nature of the addition of DNAallows for combinatorial DNA shuffling.

DNA shuffling can also be conducted on multiple genes in parallel in asingle reaction pot. For example, DNA hybridization is a discreteprocess; under stringent conditions, oligos from gene A will onlyrecognize DNA from gene A or related sequences, and ‘ignore’ oligos ofnon-gene A sequences. Assuming that gene A is unrelated to gene B, onecan mix solid supports containing oligos from gene A and gene B, and mixthem simultaneously with the DNAse treated fragments. Thus, severalgenes can be shuffled simultaneously, in the same reaction vessel.

As noted, solid phase shuffling provides several advantages. It is worthnoting certain additional advantages. For example, solid phase synthesisof nucleic acids, proteins and other relevant components isstraightforward, simplifying automation processes. Similarly, tetheringoptionally utilizes the attachment of oligos to gene chips, acommercially available technology platform (e.g., from Affymetrix, SantaClara, Calif.). One may generate gene chips for shuffling or otherdiversity generation reactions.

Further, since the addition of DNA to the tether (assembly) is stepwise,this step by step process can be controlled (i.e. the reaction can bestopped at any point and conditions changed, such as temperature, salt,extension time, etc).

One can include RNA polymerase promoters on oligos used in the assembly(i.e., an oligo 5′ to the coding region), and thereby transcribe RNA invitro from the solid support linked gene libraries. Since one cantranscribe RNA in vitro from these libraries, one can also translate invitro to directly generate libraries of proteins without cloning. Evenif yields of proteins from in vitro translation are low, the translationnonetheless allows very fast screening methods to be employed. Even lowlevels of expression are sufficient for a variety of methods such asantibody-based screening methods (e.g., ELISA) and enzyme-baseddetection assays in which signal is amplified in the assay process.

Because tethered DNA is easily purified, libraries can be pre-screenedprior to cloning, to select for certain traits, or to select againstcertain traits (for example hybridization to a gene of interest, or lackof hybridization to the gene of interest), e.g., using appropriate genechips.

Finally, the technology of using tethered molecules offers advantages inlibrary tracking and cataloging.

Methods to purify only desired shuffled genes can be employed. Forexample, it is often advantageous to purify only those shuffled genesthat are full-length (partial sequences are often less likely to beactive). For example, one can synthesize a shuffled library with atethered oligo that lies 3′ to the gene of interest, using an oligo thatincorporates a promoter for an RNA polymerase (e.g. T7 RNA polymerase)5′ to the coding region in the assembly process. RNA is transcribedusing T7 polymerase. The resulting sample is treated with nuclease thatdestroys single stranded DNA but protects RNA/DNA hybrids (for e.g. S1or Mung bean nuclease). DNA still linked to the solid support ispurified. The sample is heated, or RNAse treated to remove RNA. An oligothat anneals to sequence near the 5′ end of the gene (internal to T7polymerase promoter, but 5′ to region of interest) is hybridized. Thesingle stranded DNA product is extended using DNA polymerase to give adouble stranded product. The materials is removed from solid support andcloned, or is in vitro transcribed (in place or in another reactionvessel).

Tethering methods include: chemical tethering, biotin-mediated binding,cross-linking to the solid support matrix (e.g., U.V., or florescenceactivated cross-linking) and the use of ‘soluble’ matrix, such as PEG,which can be precipitated by ETOH or other solvents to recover boundmaterial (see Wentworth, P., 1999, TIDTECH 17:448-452).

(1.) Combinatorial Shuffling Using Solid Supports

By performing diversity generation reactions such as shuffling on solidsupports, the variation accumulated in such experiments can becontrolled. By using oligos linked to solid supports as outlined above,one can perform sequential additions of DNA by annealing and extension.

In one specific embodiment, this process is performed by: (1) for eachfamily member, PCR amplifying the region of interest, digesting withDnase, and isolating fragments. (2) Placing Dnased fragments for eachgene in a separate ‘cup’ (i.e., a cup for gene A, a cup for gene B, acup for gene C, a cup for gene D). Each cup contains DNA fragmentsrepresenting the whole of each gene, but each gene has its own cup. (3)In the first step, a single stranded oligonucleotide linked to a solidsupport, (with 10-30 bp of accessible DNA, and an exposed 3′ hydroxyl)is divided into several equal fractions (in this example 4 fractions).Each fraction is placed into a separate ‘cup’ of DNA fragments fromeither gene A, B, C, or D. The ‘cups’ are heated to denature any doublestranded hybrids present in each cup, then cooled to allow DNA toanneal. During this annealing, fragments homologous to the solidsupport-linked oligo anneal to this oligo. The annealed products arethen extended with DNA polymerase to yield double stranded product,linked to the solid support (in this example, one fourth of the DNA is a‘cup’ containing gene A sequence, one fourth in a cup containing gene Bsequence, one fourth gene 3, one fourth gene 4; however, an advantage ofthe system is that any ratios of starting genes may be used, e.g., tobias resulting recombinant nucleic acids towards one parent type).Following the ‘extension’ reaction, the double stranded DNA fragmentsare removed by virtue of their solid support linkage (for e.g. magneticbeads), and pooled into one tube (or other container). These hybrids areheated to denature the duplexes, and the unlinked strand washed away.

In a second round, the newly extended single stranded fragments areagain randomly divided into pools (in this case 4), and each portion isagain placed into one of the available cups (in this case 4 cups, forgenes A, B, C, D). The ‘cups’ are heated to denature any double strandedhybrids present in each cup, then cooled to allow DNA to anneal. Duringthis annealing, fragments homologous to the solid support-linked singlestranded polynucleotide anneal. The annealed products are then extendedwith DNA polymerase to yield double stranded product, linked to thesolid support (in this example, one fourth of the DNA was is a ‘cup’containing gene A sequence, one fourth in a cup containing gene Bsequence, one fourth gene3, one fourth gene 4). Once again the extendedproducts are removed and re-pooled into one container. This container isheated to denature the double stranded duplexes, and the strand unlinkedto the support washed away. The support-linked polynucleotide collectionis now divided once again, and the process repeated.

After a sufficient number of annealing/extension reactions, the finalsingle stranded DNA products can be converted to double stranded DNA byannealing an oligonucleotide internal to the last oligonucleotidecapable of attachment, and extended with DNA polymerase and dNTPs. Thedouble-stranded products are then released from the solid support, andcloned. In order to facilitate cloning, several rounds of PCRamplification may be performed in the tube containing the support linkedoligonucleotide, and this may act as a template for PCR while stillattached to the solid support. Cloning can also be facilitated byincorporating the recognition sequence for one or several restrictionnucleases into the sequence to be incorporated at each end of theassembled gene fragment.

One can design methods to eliminate support-linked oligos that fail toextend in any one step, if this is a source of substantial background.

(2.) Shuffling Using a Tethered Single-Stranded Template

As an alternative to tethering oligonucleotide primers to a solidsupport, single-stranded template polynucleotides can be immobilized ona solid support as described above (e.g., by: chemical tethering,biotin-mediated binding, cross-linking to the solid support matrix,etc.). In one preferred embodiment, the template polynucleotides arearrayed by depositing a solution containing the template nucleic acidson a glass slide coated with a polycationic polymer such as polylysineor polyarginine (see, e.g., U.S. Pat. Nos. 5,807,522 and 6,110,426“METHODS FOR FABRICATING MICROARRAYS OF BIOLOGICAL SAMPLES” to Brown andShalon. The template polynucleotide can be either DNA or RNA, or acombination of DNA and RNA. A wide variety of suitable templates exist,and can be selected by the practitioner depending on the specificapplication. For example, desirable template polynucleotides includegenomic and/or expressed (e.g., cDNA) sequences including coding,non-coding, antisense, naturally occurring, artificial, consensus,synthetic and/or substituted (e.g., dUTP substituted DNA) molecules. Insome applications, a population of identical polynucleotides are arrayedon a support. In other applications, templates representing a diversepopulation of polynucleotides are attached to a support. For example,entire genomes, e.g., bacterial or fungal genomes can be arranged in aphysical array on a glass slide or silicon chip. In yet otherapplications, the expression products of a cell, or a subset thereof areaffixed to the support. Such expression products can be RNA or cDNA, andin some cases comprise a library of expression products. The presentinvention is not limited by the choice of template, or the source ofpolynucleotide selected. Such routine selections are based on theparticular application, and will be readily apparent to one of skill inthe art.

Diversity is introduced by hybridizing single-stranded nucleic acidfragments to the immobilized template polynucleotide. Typically, thenucleic acid fragments will possess regions of sequence similarity (oridentity) as well as regions of dissimilarity. In many cases, annealingof multiple complementary (or partially complementary) fragments resultsin hybridization of partially overlapping fragments to the immobilizedtemplate. A polymerase (e.g., a DNA or RNA polymerase such as athermostable DNA polymerase) is used to extend the annealed primersgenerating a heteroduplex made up of the template and a substantiallyfull-length heterolog complementary (i.e., that hybridizes) to thetemplate nucleic acid. Optionally, the unhybridized overhanging regionscan be removed, e.g., with a nuclease, prior to or following extension,and/or the gaps between annealed (and extended) fragments joined with aligase. In some cases, it is desirable to employ a nuclease or ligasewith polymerase activity. This process is illustrated in FIG. 31, inwhich a solid phase-bound template is hybridized to appropriatefragments. As shown, the fragments are extended, if desired, unwantedflaps are digested and breaks in the resulting extended nucleic acidssealed with ligase.

The process can be repeated for multiple cycles by denaturing theheteroduplex and hybridizing the template to a new set (or subset) ofnucleic acid fragments. The recombinant heterologs generated in eachcycle are optionally recovered between successive cycles of denaturationand reannealing. Most typically, recovery relies on amplification,although other methods such as hybridization and/or cloning are alsofeasible. Optionally, the recovered heterolog can be used directly inadditional diversity generating procedures, as described herein and inthe cited references.

Frequently, recovery is facilitated by incorporating a sequence thatserves as a primer for the amplification reaction within the template ora fragment nucleic acid sequence. For example, the template canincorporate recognition sequences for “universal” and “reverse” primersat its 5′ and 3′ ends, respectively. Among the fragments hybridized tothe template are included the corresponding universal and reverseprimers. Subsequent amplification of recombinant polynucleotides thenproceeds according to routine amplification procedures.

In addition to the commonly used linear sequence primers (such asuniversal and reverse primers), the present invention makes use ofprimer sequences with a specialized secondary structure for facilitatingrecovery of the recombinant heterologs generated by extension offragments annealed to a specified template. For example, a boomerang DNAamplification reaction is primed by a single primer located internal torecombinant heterolog (for example, a conserved region of thetemplate/fragments can be selected for use as a primer binding site). Asillustrated in FIG. 32A, adaptors that assume a hairpin configurationare ligated to the end(s) of the heteroduplex which is optionallyreleased from the solid support. Following denaturation of theheteroduplex, and binding of the internal primer, extension by a DNApolymerase results in extension of a product including sequencesidentical to the heterolog and the template as an inverted repeat.Typically, a restriction enzyme recognition site is incorporated intothe hairpin, permitting separation of the template and heterologsequences.

Another alternative is to employ a “vectorette.” In this approach,amplification occurs between an internal primer and a primer within thevectorette, a pair of synthetic oligonucleotides having regions ofduplexed DNA flanking a central mismatched region that provides a primerbinding site, as illustrated in FIG. 32B. If the target nucleic acidsare cleaved with a restriction enzyme prior to ligation of thevectorette sequence, only restriction fragments including the internalprimer binding site are amplified. A first extension cycle results in aduplex corresponding to the recombinant heterolog which can be simplyamplified using the internal and vectorette primers. A variation of thisapproach is the “splinkerette,” in which the vectorette incorporates alooped-back hairpin structure that decreases end-repair priming andreduces non-specific priming. Further details on vectorette use andconstruction can be found in Arnold et al. (1991) “Vectorette PCR: anovel approach to genomic walking” PCR methods Appl. 1:39-42 and Hengen(1995) “Vectorette, splinkerette and boomerang DNA amplification” TrendsBiochem Sci. 20:372-3.

As previously described, recombinant nucleic acids produced byhybridization and extension of nucleic acids on an array can further betranslated to provide reaction products suitable for screening.Alternatively, the recombinant heterologs described above can betransformed and expressed in cells to facilitate screening by structuraland/or functional means to identify recombinants with desirableproperties. Typically, but not necessarily, the recombinant nucleicacids are introduced into host cells in a vector, such as an expressionvector. Vectors and cells incorporating recombinant polynucleotidesproduced by the above described recombination on a solid phase supportare also a feature of the invention.

H. An Example Integrated System for Diversity Generation Via Shuffling

This example “shuffling machine” is an integrated system which convertsparent DNA into improved shuffled clones, which are optionally used asparent DNAs for subsequent shuffling. The machine is based upon a set ofmodules as discussed above that are integrated to improve function andthroughput.

The machine performs a number of tasks, using a liquid handling station,a PCR system, a fluorescence/absorbance plate reader, a plate/reservoirstorage device and a robotic system for shuttling plates between themodules. This machine performs the entire shuffling processautomatically in a microtiter plate format.

For clarity of description, the machine is split into a number ofmodules; however, module functions can be combined in practice tosimplify the overall system. An example schematic of the modules of anintegrated shuffling machine is provided by FIG. 2. The modules includea shuffling module, a library quality assessment module, a dilutionmodule, a protein expression module, and an assay module. Typicalintegrated device elements include thermocyclic components, single andmulti-well liquid handling, plate readers and plate handlers.

(1.) The Shuffling Module

This example shuffling module uses a liquid handler, a PCR machine, afluorescent plate reader, and a plate/reservoir handling and storagesystem to perform an automated shuffling reaction (as noted, shufflingis one preferred diversity generation reaction performed by the methodsand systems herein).

FIG. 3 provides a schematic representation of the steps performed bythis exemplar shuffling module. In particular, a single pot reaction isperformed, utilizing uracil incorporation, DNA fragmentation andassembly. A rescue PCR is performed, the results assessed with PicoGreenand any wells that test positive for PicoGreen incorporation are rescuedand sent to the library quality modules.

As noted, DNA fragmentation is achieved using the uracil incorporationstrategy noted above. Different wells of a microtiter plate are set upwith different reaction conditions, leading to different DNA sizefragments and different ratios of parental nucleic acids (the diversitytarget sequences). The conditions for the uracil fragmentation isdefined by the user as are the assembly and rescue protocols.

In other embodiments, the conditions and/or protocols are calculatedusing a set of computer understandable instructions, e.g., embodied in acomputer or web page operably coupled to the shuffling module.Alternatively, the shuffling module is optionally a programmable orprogrammed module that calculates appropriate conditions, e.g., based onempirical data, theoretical predictions and/or user input.

Once the fragmentation is complete (as selected by the user) thefragmented DNA is transferred to a PCR module for the assembly reaction.An aliquot of the assembled DNA is then transferred to a new PCR platefor a rescue PCR reaction using standard primers.

The success of the shuffling reactions are measured by removing analiquot from the rescue PCR plate and followed by transfer to a platecontaining Pico green dye.

Wells that contain double stranded DNA (i.e., give fluorescence withPico Green) are collated by the liquid handler, using hit pick software,into plate(s) that contain all the shuffled clones, which are passed onto the library quality module.

The liquid handler then transfers (and, optionally, mixes or otherwisemodifies materials) to make up solutions from solvent/reagentreservoirs, setting out an array of reactants. The information as towhich solutions are plated in which positions in an array is trackedthrough subsequent manipulations in all modules, along with the PCRconditions which are used for amplification.

Once the rescue PCR is performed, the success of the recombination isassigned based upon the presence of double stranded DNA as measured byPico Green fluorescence. Full length ds DNA can also be unambiguouslyidentified and quantified by capillary electrophoresis (e.g., inparallel formats similar to a parallel capillary electrophoresissequencer such as MEGABASE or by parallel capillary electrophoresis on achip) with detection by fluorescence. Successful recombination leads topredominantly a single full-length species in the rescue PCR which isproportional to an arbitrary level of fluorescence. As noted above, Picogreen is a quantitative measure of the amount of ds DNA present and thisinformation about the DNA concentration in each well is used in thedownstream processing modules. The hit picking software takes thepositive wells and converts them to new well positions without loss ofinformation. The set of positive wells across all of the plates isreferred to as a “collated library.”

Another exemplary shuffling module or diversity generation devicecomprises a programmed thermocycler and fragmentation module operablycoupled to the thermocycler. The programmed thermocycler typicallycomprises a thermocycler operably coupled to a computer comprising oneor more instruction set. In other embodiments, the instruction sets areembodied in a web page or in the thermocycler itself, e.g., a Javaprogram. For example, a network card is optionally added to athermocycler or the internal software of a commercially availablethermocycler is altered to provide the instruction sets described below.

The instruction sets typically comprise computer understandableinstructions for performing one or more of the following: calculation ofan amount of uracil and an amount of thymidine for use in the programmedthermocycler; calculation of one or more crossover region between two ormore parental nucleotides; calculation of an annealing temperature;calculation of an extension temperature; and/or selection of one or moreparental nucleic acid sequence. These calculations are typically madebased on one or more of: user input, empirical data, and theoreticalpredictions, e.g., of melting temperature. Such melting temperaturepredictions are well known to those of skill in the art. In addition,predictions are also optionally used to calculate the effect ofannealing temperatures on the number of possible crossovers. Typicalinput data include, but are not limited to, parental nucleic acidsequences, desired fragmentation lengths, crossover lengths, extensiontemperatures, and annealing temperatures. Empirical data typicallycomprise comparisons of one or more nucleic acid melting curve ormelting temperature.

The computer or programmable thermocycler typically calculates possiblecrossover regions between parental nucleic acid sequences, depending onthe annealing temperature and extension temperatures to be used in theamplification steps. The computer would then set up one or more cyclefor the thermocycler. For example, a cycle in the thermocycler typicallyincludes amplification of one or more parental nucleic acid sequence,fragmentation of the one or more parental nucleic acid sequence toproduce one or more nucleic acid fragments; reassembly of the one ormore nucleic acid fragment to produce one or more shuffled nucleic acid;and, amplification of the one or more shuffled nucleic acid. Variousrobotics and plate handlers are optionally added to the device asdescribed herein to transfer nucleic acids between the fragmentationmodule and the thermocycler.

In some embodiments, the thermocycler amplifies the various parentalnucleic acids in the presence of uracil and the fragmentation devicefragments the parental nucleic acids using various uracil cleavingenzymes. The programmable thermocycler in this embodiment typicallydirects a pause in the cycle to allow the addition of the enzymes to thereaction mixtures. In addition, the programmed thermocycler is used tocalculate the ratio of uracil residues to thymidine residues to producefragments of a desired mean length or size. For example, a length thatleads to an optimized level of diversity in the shuffled nucleic acidsis optionally selected. Fragmentation is optionally carried out in thepresence of Taq/Pwo and outside primers so that the fragments are useddirectly in the reassembly/amplification steps of the cycle withappropriately calculated annealing and extension temperatures. Otherfragmentation methods optionally used in a fragmentation module of theinvention and operably coupled to a programmed thermocycler include, butare not limited to, sonication, DNase II digestion, random primerextension, and the like.

In another embodiment, a diversity generation device comprises acomputer, a synthesizer module, e.g., a microarray oligonucleotidesynthesizer such as an ink-jet printer head based oligonucleotidesynthesizer, and a thermocycler. The computer typically comprises atleast a first instruction set for creating one or more nucleic acidfragment sequence from one or more parental nucleic acid sequence. Thesynthesizer module typically synthesizes the one or more nucleic acidfragment sequence created by the computer; and the thermocyclergenerates one or more diverse sequence from the one or more nucleic acidfragment sequence, e.g., by performing an assembly/rescue PCR reactionas described above. For example, the synthesizer optionally synthesizesthe nucleic acids fragments on a solid support as described above, e.g.,using mononucleotide coupling reactions or trinucleotide couplingreactions.

In addition, the computer optionally comprises additional instructionsets, e.g., for determining a set of conditions for the thermocycler,e.g., to perform assembly/rescue PCR reactions.

For example, sequences, e.g., DNA, RNA, or protein sequences, areentered into a computer, e.g., character strings corresponding to thesequences. The computer is then used to generate a number of smallersequences from which oligonucleotides can be created. These smallersequences typically encode for some or all of the diversity of theoriginal sequences entered. Typically, the instruction sets, e.g., in acomputer, or web page, or both, limit or expand diversity of the one ormore nucleic acid fragment sequence, e.g., a parental nucleic acidsequence, by adding or removing one or more amino acid having similardiversity; selecting a frequently used amino acid at one or morespecific position; using one or more sequence activity calculation;using a calculated overlap with one or more additional oligonucleotide;based on an amount of degeneracy, or based on a melting temperature. Thesequences are then used to drive a synthesizer, e.g., an oligonucleotidesynthesizer, to create a physical manifestation of the sequences, e.g.,on a support medium or solid support. Once the oligonucleotides aresynthesized, the solid support is optionally digested or theoligonucleotides are cleaved from the support, e.g., using thethermocycler. The mix of oligonucleotides is then used in thethermocycler, which creates full length sequences, e.g., shuffledsequences. The computer is also optionally used to determine the bestconditions for assembly/rescue reaction and digestion.

The above device allows one to generate synthetic shuffled genesstarting with only sequence data in a matter of hours. Combined with ahigh throughput screening device the genes are all optionally createdand screened for desired characteristics in less than a day. Therefore,the devices described above also optionally comprise screening modules,e.g., high-throughput screening modules, for screening the one or morediverse sequence for a desired characteristic. In addition, the computeris optionally used to select the original sequences used to create thefragments for shuffling, as described above.

The above diversity generation devices are typically used to allow rapidshuffling of nucleic acids to create new and diverse nucleic acids,e.g., enzymes. In some embodiments, the devices are incorporated intokits comprising, e.g., the devices, reagents, and appropriate protocolsfor shuffling. For example, a kit optionally comprises a diversitygeneration device as described herein, e.g., comprising a pre-programmedPCR machine, and one or more reagent for generating diverse nucleicacids. Reagents include, but are not limited to, E. coli, e.g., adut-ung strain to make plasmids containing uracil instead of thymidine,PCR reaction mixtures comprising a mixture of uracil and thymidine, oneor more uracil cleaving enzyme, a PCR reaction mixture comprisingstandard dNTPs, polymerases, and the like. Possible uracil cleavingenzymes included in the kit are uracil glycosidase, an endonuclease,such as endonuclease IV, and the like. The uracil/thymidine ratiosincluded with the kit can be optimized to produce fragments ofparticular size or the protocols and/or diversity generation devices areprogrammed to calculate the appropriate ratios. Concentrations of dNTPs,Mg and other reagents are also optionally provided in optimized formats.In addition, the number of cycles is also optionally optimized, e.g., bya programmed thermocycler.

Polymerases included with the kits are typically thermostablepolymerases, e.g., non-proof reading and proof-reading polymerases. Inaddition, the kits optionally include artificially evolved enzymes,e.g., artificially evolved polymerases that have a higher fidelity ofincorporation for uracil residues, or are more active at 25° C. thanthose presently available.

The kits and devices above are optionally used to create en entirelyautomated format for generating diversity, e.g., through shuffling. Inaddition, they can be combined in a variety of ways with othercomponents described herein, e.g., to create high throughput shufflingand screening capacity.

(2.) Library Quality Module

The library quality module utilizes the liquid handler, the PCR system,the Fluorescence Plate Reader and the Plate/reservoir handling andstorage system.

FIG. 4 provides a schematic overview of a Library Quality Module. Inparticular, the module divides reactions into multiple plates, performsa crossover assessment, verifies PCR by PicoGreen incorporation andperforms a hit pick quality rating.

The collated shuffled library from the shuffling module are diluted intoone or more daughter plate to achieve a standard DNA concentration. Thisdaughter plate is used as the source plate for DNA templates in qualityassessment PCR reactions. Each parental DNA serves as the template todesign forward and reverse PCR primers. These primers are mixedcombinatorially such that recombinants can be detected (e.g., by mixingforward primer “A” which uniquely recognizes parent “A” with reverseprimer “B” which uniquely recognizes parent “B,” etc., covering allpossible combinations of primers, or a desired subset thereof). The PCRreactions are transferred to a plate for Pico Green quantitation. Thecollated libraries are ranked with respect to diversity based on thelevel of fluorescence in each reaction and the number of PCR reactionsthat give amplification. The top collated libraries are then(optionally) re-collated to provide diverse collated libraries which arepassed onto the in vitro transcription/translation module, or the hitsare simply passed onto the in vitro transcription/translation module.

The DNA concentrations determined by the shuffling module is used tonormalize template DNA concentrations in this module. The number ofdifferent PCR reactions run is determined by the number of startingparental sequences and the amount of information desired (e.g.,2^(no of parents−1) reactions gives good information) to determine thebest library. An hypothetical “perfect” library gives the sameamplification rate (and hence fluorescence) in each PCR reaction. Whilethis does not give the number of crossover genes per se, it can be usedto ensure that the there is a diversity of sequences that have at leastone crossover.

(3.) Dilution Module

The dilution module uses the liquid handler, the PCR system, thefluorescence plate reader and the plate reservoir handling/storagesystem.

FIG. 5 provides a schematic overview of the dilution module activities.In particular, DNAs are diluted to the desired number of copies perwell, PCR amplified, assessed for dsDNA by PicoGreen, and hits arepicked.

The top collated libraries are reamplified, incorporating a reporterprotein into the library, either as a fusion or as part of atranslationally coupled system. An aliquot of this material is removedfor quantitation and the library is diluted and dispensed intomicrotiter wells at an average concentration of about 1-10 DNAmolecules/well.

The DNA is amplified by PCR to give enough DNA for efficient in vitrotranscription/translation (ivTT) and an aliquot is removed forquantitation with Pico Green. The wells where DNA is amplified are thenhit picked into wells ready for transfer to the protein expressionmodule. A number of wells in each plate are filled with standard controlconstructs (e.g., wild type and a negative control) at the sameconcentration as the library clone pools.

In general, the dilution which gives a concentration of 1-10 DNAmolecules/well is determined from a standard curve. The reporter proteinis chosen to give a construct that efficiently undergoes ivTT for alarge number of systems. This also standardizes the ivTT procedure forall proteins.

(4.) Protein Expression Module

The Protein expression module uses the liquid handler, the fluorescentplate reader and the plate/reservoir handling and storage system.

FIG. 6 provides a schematic overview of the activities of the expressionmodule, i.e., the addition of DNA to cell-free ivTT reaction mixtures toform arrays of reaction mixtures, an assay for a co-translationalproduct as a control, and the picking of hits by the presence of theco-translational control product.

The pooled library members are taken from the dilution module and analiquot is removed in which the DNA concentration is adjusted foroptimal ivTT. The rest of the ivTT mix is then added to the wells andprotein production is initiated. The efficiency of the ivTT reaction ismeasured using the activity of the reporter protein. For example, if thereporter is green fluorescence protein (GFP), then efficiency ismeasured by directly monitoring fluorescence. If the reporter is anenzyme, an aliquot is typically removed for appropriate processing.

The wells which give efficient protein production are then rearrayedinto new microtiter plates and passed on to the assay module.

The DNA concentration in each well is determined by the dilution moduleand therefore the amount of DNA in each well can be normalized to acorrected value for efficient ivTT. The wells which contain the controlconstructs are tracked so that the activity of the library clones can becompared to the initial wild type.

(5.) Assay Module

The Assay module uses the liquid handler, afluorescent/colorimetric/luminometer plate reader and theplate/reservoir handling and storage system.

FIG. 7 provides a schematic overview of the exemplar assay module. Inparticular, expression mixtures are added to assay reagents (or viceversa) and changes in a detectable marker such as absorbance,fluorescence or luminescence are detected and hits picked. Similarly,the assay module can include an autosampler which interfaces with a CE,MS, GE or other system. SPR (surface plasmon Resonance) can also be usedto measure protein binding. SPA (Signal Proximity Assay) methods canalso be used, e.g., using a luminescence plate reader.

The protein solutions provided by the protein expression module aretested for the properties of interest. The proteins are typicallydiluted to a standard concentration before the assay, using the level ofthe reporter protein as a marker.

The protein solutions are aliquoted out and assayed using any formatthat leads to a spectrophotometric change in the properties of the assaymix. A majority of proteins may be assayed, directly or indirectly,using such formats (e.g., to monitor changes in pH, production offluorescent product, loss of turbidity on hydrolysis, coupled assays,etc.).

Alternatively, the proteins can be assayed using heat production oroxygen consumption, changes in conductivity (ion production), parallelCE, GC, or the like. These properties of solution are readilyquantified, e.g., using microfabricated devices as discussed above.

The proteins that are determined to be better than wild-type accordingto the criteria of the assay are identified and the position of theclones are determined.

The proteins are normalized to account for expression artifacts in theivTT reaction. The activity of both the wild type and negative controlclones is measured and used as a measure of the range of the assay. Thevariation in the controls (standard deviation) determines howsignificant differences are among the hits, as well as providing forstatistical comparisons (e.g., standard average deviations as comparedto wild type, etc.).

(6.) Deconvolution of Hits and Retesting

The clone pools can be reconfirmed and deconvoluted by submitting themto the dilution module. This separated the pool of about 10 clones intoa few hundred wells, with increased stringency (to about 1molecule/clone per well). The remaining modules then retest eachmolecule one or more times, verifying the previously identifiedactivity. The assay module can also incorporated a secondary assay tofurther verify desired activities.

(7.) Second Round Shuffling

The reconfirmed hits are optionally used as substrates in subsequentshuffling reactions, with this process being iteratively (andautomatically) repeated by the various modules of the system, until adesired activity level for the target is obtained.

(8.) Example Machine Configuration

FIG. 8 provides an exemplar configuration for a recombination andselection machine, showing plate stacker 801, gantry robot 805,pipetting heads 807, plate gripper 809, plate reader 811, thermocycler813, plate holders 815, solution reservoirs 817 and reagent tubes 819.During operation of the device, plates are transferred from platestacker 801 by plate gripper 809 to plate holders 815 to the variousoperation regions such as thermocycler 813 and plate reader 811. Platesare also optionally transferred back to plate stacker 801. Reagents aretransferred to and from reagent tubes 819 and solution reservoirs 817via pipetting heads 807, which also transfer materials between reagenttubes 819, solution reservoirs 817 and any plates used in the system.

(9.) Example Miniature Configuration

In this example, a miniature laboratory system is used, e.g., to performa shuffling reaction. As shown in FIG. 19, the system includes anappliance and a microfluidic chip which has environmental control layer19-1, microfluidics layer 19-2 and support layer 19-3, as well asoptical interface for temperature control 19-4 and power supply 19-5(see also, FIGS. 20-22). In operation, the miniature laboratory systemis used, e.g., in combination with a module that provides reagents andoptimal environmental conditions. Starting materials that are providedinclude DNA (genes/gene fragments, oligonucleotides, etc.), reagents,primers vectors, etc. The product of the system is, e.g., a gene libraryof diversified genes, operons, etc. Additional steps can be included inthe system for additional reactions, if needed. Where purification stepsare desired, membrane filters are optionally positioned in the flowlines, e.g., binding reagents or components that are to be removed. Themicrofluidics system that is used in the miniature laboratory system isused to guide and direct low volume samples containing, e.g., 0.05-100ng/μl of DNA. Using advanced separation systems and DNA reactionchambers, DNA shuffling can be performed in the miniature laboratorysystem.

As shown in FIG. 19, in one embodiment, a three-layer chip constructionis used to provide the microfluidic portion of the overall system. Thebottom layer is for support, the middle layer contains channels thatguide DNA and solutions and reagent solutions and the top layer providescontact points for a power supply and a temperature controller (e.g.,operating by conductivity or light). Details regarding the top layer arefound in FIG. 20. Samples are transported through the system, e.g., byair pulses or other fluid driving means. Details regarding the fluidicslayer is set forth in FIG. 21. An appliance (FIG. 22) contains theoperation hardware (and optionally software) for the miniaturelaboratory system, including PCR programs, incubation periods, DNAseparation and sample product import/export. The appliance alsooptionally interfaces with a computer to provide additional controlfeatures. The complete system provides means to generate libraries ofshuffled genes directly, by supplying starting DNA, reagents,oligonucleotide primers and vectors. The resulting DNA sample isdirectly introduced into, e.g., a cell of choice by transformation,electroporation, conjugation, particle bombardment, injection, etc.

I. Example DNA Shuffling Machine (Alternate Embodiment)—Comparison ofAlternate Breeding Strategies

One way to develop more sophisticated breeding strategies is toempirically compare different breeding strategies. A DNA shufflingmachine allows for increased throughput and accuracy in molecularmatings.

Standard DNA shuffling is done, e.g., by purifying DNA fragments ongels, assembling fragments in a PCR machine, rescuing fragments in a PCRmachine, and then cloning the final rescued product. The essentialconstraint with this approach is that it requires skilled labor and itis typically costly for a given person to sample a more than a fewshuffling variables. However, there are many variables of interest, suchas pairwise vs. pooled matings, fragment size, stoichiometry of theparental genes, degree of random mutation vs generating diversity byrecombination, etc.

This example provides a solution to this difficulty by automating theshuffling process, providing scalability and other advantages. Theexample DNA shuffling machine which is the subject of this example isembodied in FIGS. 10 (showing a schematic of the DNA shuffling machine),11 (showing a schematic of a DNA fragmentation device), 12 (showing aschematic of a DNA fragment analysis and isolation device), 13 (showinga schematic of a DNA fragment preparation device), 14 (showing aschematic of a precision microamplifier), 15 (showing a schematic of aDNA assembly and rescue module), 16 (showing a schematic of arecombination analysis device), and 17 (showing a schematic of arecombination analysis device).

FIG. 10 describes an overall DNA shuffling machine (10-1). Thisdevice/system can be built either as an integrated unit, or as aseparate module. It can be designed to handle multiple samples inparallel, as each of the modules is scalable. As shown, Input elementsincluding, e.g., plasmids, PCR products, genomic DNAs, primers, etc. arefragmented in DNA fragmentation device or module 10-2. Also included areDNA assembly and rescue device or module 10-3 providing for outputs,e.g., in the form of recombined/shuffled inserts. Finally, recombinationand analysis module or device 10-4 provides for recombination analysison any recombined/shuffled materials (e.g., shuffled insert DNAs).

FIG. 11 describes a DNA fragmentation device. For the purpose ofautomation, a reliable, preparation independent method to producefragments of a desired size is useful. Sonication is a useful methodbecause the fragment length depends on purely physical parameters suchas the frequency of sonication and the viscosity of the fluid. However,one issue with this method is the type of ends that are generated, as 3′hydroxyl ends are preferred for subsequent assembly steps to work. Theaddition of chemical cleaving agents can improve the yield of 3′hydroxyls in the sonication reaction. Enzymatic treatment with anuclease that is specific for, for example, 3′ phosphates, improves thequality of sonicated fragments for DNA shuffling reactions. Otherfragmentation methods discussed supra can also be adapted to the presentexample, such as the use of point-sink shearing methods, synthesis, etc.

FIG. 12 describes a DNA fragment analysis and isolation device. Acapillary electrophoresis instrument (e.g., column 12-1) is used toseparate the DNA fragments. A detector monitors fluorescently labeledmarkers on the column to a “waste” or to “collection” reservoir. Thisallows for automated collection of DNA fragments in the size range thatis programmed by the user. An analytical instrument, made of componentssimilar to those used for sequencing gels, can be used for theanalytical runs for doing analysis of PCR with recombination oligos orfor analysis of raw assemblies to assess the efficiency of assembly. Forexample, one can collect 25-50 bp fragments.

FIG. 13 describes a DNA fragment prep device. The DNA is denatured toexpose or create single stranded DNA that binds efficiently to a C18hydrophobic column and which can be quantitatively eluted andconcentrated. This uses the principle of the SEP-PAK C18 column, but ismodified for use in an automated device. Alternatives to this approachinclude ion exchange chromatography, precipitation, lyophilization, etc.

FIG. 14 describes a precision microamplifier (PMA). DNA 14-6 is placedin microcapillary 14-7 between two drops of oil (14-4, 14-5) to seal itagainst evaporation. Typical drop sizes range from 1 nl to 1 μl. Themicro-capillary is moved through three resistors (14-1, 14-2, 14-3)whose temperatures are programmed. As depicted, robotic arm 14-8 is usedto move the capillary, and thus the DNA droplet, e.g., between resistors14-1, 14-2, and 14-3. In the simplest case, the resistors are set for,e.g., 93, 45 and 72 degrees centigrade. By moving cyclically throughthese temperatures, a PCR or assembly reaction can be driven inmicrodroplet in the microcapillary. A chief advantage of this relativeto a standard PCR machine is that the temperature can be controlled moreprecisely, and, more importantly for DNA shuffling, the volume of theassembly reaction can be driven into the submicroliter range veryeasily. This allows shuffling using small quantities of fragments,allowing for more molecular “crosses” in the shuffling reactions from agive amount of input DNA.

FIG. 15 describes DNA assembly and rescue module 15-1. Assembly is donein a modified PCR machine or in the PMA (depicted as assembler 15-2).The PMA, or similar low-volume/high throughput methods provide onepreferred approach, because one can amplify very small volumes whichprovides for shuffling using a smaller quantity of fragmented DNA. TheAnalyzer provides a quantitative way to monitor the size anddistribution of PCR products and the properties of PCR rescue. A cleanand efficient rescue of a unit length of a gene fragment is preferred.The size distribution of assembled product and the properties of therescue PCR are highly informative for predicting the efficiency ofshuffling that has occurred. The analysis can be done by capillaryelectrophoresis or by mass spec. As depicted, various inputs, includingrandom DNA fragments, overlapping PCR fragments and the like areassembled in assembler 15-2. The assembly and rescue module furtherincludes rescue PCR element 15-3 and analyzer 15-4 (e.g., including acapillary electrophoresis module). Assembly module 15-1 produces outputsincluding assembled fragments, rescued PCR inserts and the like.Analyzer 15-4 provides profile information including size distributioninformation.

FIG. 16 describes recombination analysis device/module 16-1. Inputsinclude raw assembled components and PCR rescued assembled components.Outputs include analysis of the ratio of recombined to parentalsequences. In the device, “Crossover oligos” prime one or anotherparents exclusively, and thus, a 5′ oligo from P1 and a 3′ oligo from P2only PCR amplify a recombinant such as F1(B). The analyzer is, forexample, a capillary electrophoresis machine that precisely measures thesize and intensity of each band. By using multiple fluorophores in thecrossover oligos, one can measure, e.g., all four PCR products of theamplification in a single lane, if desired. In the figure, P1=parent #1;P2=parent #2; F1(A) and F1(B) are recombinants with structures withrespect to the crossover oligos as shown. The crossover oligos are setsof oligos that exclusively (or at least preferentially) prime theindicated parents. The strategy can be generalized to accommodatemultiple pairs of crossover oligos. An advantage of the recombinationanalysis device is that it allows one to quantitatively monitor theshuffling reaction. For example, if 100-200 base fragments are used inthe shuffling, then crossover oligos that are 300 bp apart in theassembled genes are almost fully recombined (recombinants F1(A) andF1(B) bands of only half the intensity of the parental bands.

The DNA fragmentation device and the DNA Fragment Prep Device take thetedium out of preparing gene fragments. They can also increase the yieldof fragments of the desired size. The assembly and rescue device allowsone to test multiple assembly conditions; e.g., if the precisionmicroamplifier is used for the assembly. The analysis instrument allowsone to quantitatively monitor the growth of the shuffled product. Thisanalysis capability is useful for trouble shooting, which ultimatelymakes the process even more predictably automatable.

The recombination/analysis device allows one to quantitatively measurethe frequency of recombination between any known DNA polymorphisms inthe parental genes. This analysis is useful in the optimization ofshuffling reactions generally. It is similar in effect to measuringrecombination frequencies in populations. Importantly, it allows one tomake an educated decision as to whether a given shuffling reaction isworth cloning, or in vitro expressing and screening in functionalassays, as opposed to doing further work to optimize the shufflingreaction to get a desired spectrum of recombinants. This is ofparticular value when the number of clones that can be screened islimited or costly.

J. Example: Establishment and Automated Processing of Expression Arraysfor Nucleic Acids Derived from a Variety of Sources.

Identification and characterization of genes from macro- andmicro-organisms, enrichment cultures, fermentation broths anduncharacterized environmental isolates, and the like is of commercialvalue. These genes can be used as substrates in the various diversitygeneration reactions herein. Various approaches for using diversesources of materials in the systems of the present invention areschematically outlined in FIGS. 23-30.

In the process embodiment of FIG. 23, nucleic acids are sourced from anyof a variety of diverse sources, including any of those listed in thefigure (humans and other vertebrates, other eukaryotes, oligonucleotidesand gene synthesis, etc.) The nucleic acids are extracted and/or pooled.Optionally, the pooled nucleic acids are cloned, selected, hybridized,sized, etc. The nucleic acids are then arrayed. The arrayed nucleicacids are then optionally cloned, selected, hybridized, amplified, etc.The arrays are replicated, transcribed and/or translated. The genes canbe encapsulated if desired. Proteins or bioactive RNAs are screened foractivities of interest. Finally, a physical or logical linkage betweenthe array members and the relevant observed phenotypes is established.

In the process embodiment of FIG. 24, nucleic acids are sourced from anyavailable source, including one or more of those listed in the figure,and extracted/pooled. Nucleic acids are treated with one or more enzyme,ligated into one or more vectors and introduced into cells. Cells arepropagated in the cells. Optionally, the cells or expressed nucleicacids can initially be arrayed. Clones of interest are selected using aplurality of screens, such as hybridization, complementation, etc. Theselected nucleic acids are arrayed and the arrays replicated. One ormore of the replicated arrays is transcribed and/or translated.Optionally, other arrays or array members can be cloned, selected,hybridized, etc. Bioactive RNAs or proteins are selected for one or moreactivity and, again, a physical or logical linkage between the arraymembers and the relevant observed phenotypes is established.

In the process embodiment of FIG. 25, the sourced nucleic acids (again,from any of a variety of diverse sources, including any of those listedin the figure) are extracted and/or pooled, hybridized with at least onesynthetic or naturally occurring nucleic acid or population from anothersource, and treated with at least one enzyme including at least onepolymerase or ligase activity. Nucleic acids are arrayed and arraysreplicated. Optionally, the arrays or array members include any of avariety of additional operations, including cloning, selection,hybridization, etc. Bioactive RNAs or proteins are selected for one ormore activity and, again, a physical or logical linkage between thearray members and the relevant observed phenotypes is established.

In the process embodiment of FIG. 26, sourced nucleic acids (also fromany of a variety of diverse sources, including any of those listed inthe figure) are extracted and/or pooled. The resulting nucleic acids arehybridized with at least one synthetic or naturally occurring nucleicacid or population from another source. The resulting hybridizationmixture is treated with at least on enzyme containing at least onepolymerase and/or ligase activity. The resulting nucleic acids areligated into a vector, introduced into cells and propagated. Optionallyan initial array of the resulting library is performed at this stage ofthe overall process. Library members (clones) are selected using one ormore screens. The selected members are arrayed and the arraysreplicated. Bioactive RNAs or proteins are selected for one or moreactivity and, again, a physical or logical linkage between the arraymembers and the relevant observed phenotypes is established.

In the process embodiment of FIG. 27, nucleic acids are sourced from anyof a variety of diverse sources, including any of those listed in thefigure (humans and other vertebrates, other eukaryotes, oligonucleotidesand gene synthesis, etc.) The nucleic acids are extracted and/or pooled.Optionally, the pooled nucleic acids are cloned, selected, hybridized,sized, etc. The nucleic acids are then arrayed. The arrayed nucleicacids are then optionally cloned, selected, hybridized, amplified, etc.The arrays are replicated, transcribed and/or translated. The genes canbe encapsulated if desired. Proteins or bioactive RNAs are screened foractivities of interest. In this embodiment, the properties which arescreened include fluorescent or luminescent properties of a particlesuch as a cell, encapsulated mixture or other matrix, liposome ormembrane encapsulated material which incorporates a viral coat protein,or other encapsulated material. The cell or other encapsulated materialis used to decide the end locations of such particles on an arraycomprising at least two designated end locations or chambers. Detectionis via FACS, microFACS (with or without a fluorescent signal),fluorescence, visible scanning, transmission or confocal microscopy,digital or high-density signal imaging, thermography, liquidchromatography, combinations thereof, or the like. A physical or logicallinkage between the array members and the relevant observed phenotypesis then established.

In the process embodiment of FIG. 28, nucleic acids are sourced from anyof a variety of diverse sources, including any of those listed in thefigure (humans and other vertebrates, other eukaryotes, oligonucleotidesand gene synthesis, etc.) The nucleic acids are extracted and/or pooled.Optionally, the pooled nucleic acids are cloned, selected, hybridized,sized, etc. The nucleic acids are then arrayed. The arrayed nucleicacids are then optionally cloned, selected, hybridized, amplified, etc.The arrays are replicated, transcribed and/or translated. The genes canbe encapsulated if desired. Proteins or bioactive RNAs are screened foractivities of interest. In this embodiment, the screening comprisescombination screening of the proteins or bioactive RNAs. Propertieswhich are screened include fluorescent or luminescent properties of aparticle such as a cell, encapsulated mixture or other matrix, liposomeor membrane encapsulated material which incorporates a viral coatprotein, or other encapsulated material. The cell or other encapsulatedmaterial is used to decide the end locations of such particles on anarray, e.g., comprising at least two designated end locations orchambers. Detection is via FACS, microFACS (with or without afluorescent signal), fluorescence, visible scanning, transmission orconfocal microscopy, digital or high-density signal imaging,thermography, liquid chromatography, combinations thereof, or the like.In addition, the array, e.g., at least one of the end locations,comprises a population of target cells in which a given biologicalactivity is directly assessed, such as cytocidal or antibioticactivities, stimulation or suppression of growth, generation of adetectable signal, or the like. A physical or logical linkage betweenthe array members and the relevant observed phenotypes is thenestablished.

In the process embodiment of FIG. 29, nucleic acids are sourced from anyof a variety of diverse sources, including any of those listed in thefigure (humans and other vertebrates, other eukaryotes, oligonucleotidesand gene synthesis, etc.). The nucleic acids are extracted and/orpooled. Optionally, the pooled nucleic acids are cloned, selected,hybridized, sized, etc. The nucleic acids are then arrayed. The arrayednucleic acids are then optionally cloned, selected, hybridized,amplified, etc. The arrays are replicated, transcribed and/ortranslated. The array members are also encapsulated in this embodiment.Proteins or bioactive RNAs are screened for activities of interest. Inthis embodiment, the properties which are screened can includefluorescent or luminescent properties of a particle, encapsulatedmixture, liposome, or mixture encased in a membrane comprising one ormore viral coat proteins which are used to decide, e.g., end locationsof such particles on an array, e.g., comprising at least two designatedend locations or chambers. Such methods include any combination of FACSor microFACS (with of without a fluorescent signal); fluorescent,visible, scanning, transmission and confocal microscopy; digital or highdensity digital imaging, thermography, liquid chromatography, and thelike. A physical or logical linkage between the array members and therelevant observed phenotypes is then established.

In the process embodiment of FIG. 30, nucleic acids are sourced from anyof a variety of diverse sources, including any of those listed in thefigure (humans and other vertebrates, other eukaryotes, oligonucleotidesand gene synthesis, etc.). The nucleic acids are extracted and/orpooled. Optionally, the pooled nucleic acids are cloned, selected,hybridized, sized, etc. The nucleic acids are then arrayed. The arrayednucleic acids are then optionally cloned, selected, hybridized,amplified, etc. The arrays are replicated, transcribed and/ortranslated. The genes can be encapsulated if desired. Proteins orbioactive RNAs are screened for activities of interest. In thisembodiment, the screening comprises combination screening of theproteins or bioactive RNAs. Properties which are screened includefluorescent or luminescent properties of a particle such as a cell,encapsulated mixture or other matrix, liposome or membrane encapsulatedmaterial which incorporates a viral coat protein, or other encapsulatedmaterial. The cell or other encapsulated material is used to decide theend locations of such particles on an array, e.g., comprising at leasttwo designated end locations or chambers. Detection is via FACS,microFACS (with or without a fluorescent signal), fluorescence, visiblescanning, transmission or confocal microscopy, digital or high-densitysignal imaging, thermography, liquid chromatography, combinationsthereof, or the like. In addition, the array, e.g., at least one of theend locations, comprises a population of target cells in which a givenbiological activity is directly assessed, such as cytocidal orantibiotic activities, stimulation or suppression of growth, generationof a detectable signal, or the like. A physical or logical linkagebetween the array members and the relevant observed phenotypes is thenestablished.

The field of gene isolation is well developed, e.g., in the expressionarray (e.g., Gene chip™, Aflymetrix, Santa Clara, Calif.) and eukaryoticgenomics areas, in which, e.g., RNA or genomic DNA is used to detect orsequence novel open reading frames. While tools for sequencing complexgenomes of higher organisms has advanced rapidly, less work has beendone on sequencing, deconvoluting or otherwise characterizing thegenetic properties of microorganisms and microbial systems. Furthermore,while the generation and use of hybridization and sequencing arrays hasundergone significant advancement, much of the advances are based on theability to identify and purify the messenger RNA or intact high MWgenomic DNA from higher organisms.

For eukaryotic mRNA, the presence of poly-adenylated tail allows rapidcreation and use of convenient EST (expressed sequence tagged)libraries. Since lower organism rarely exhibit such tails, other toolsare used for rapid cloning, characterization and analysis.

Recently, methods for extracting nucleic acids at high yield frommicrobial cultures, broths, pathogen and environmental samples have beendescribed. Where complex, soil-containing or mixed culture systems aretargeted for characterization or gene mining, these methods generallyuse any of a variety of treatments to provide high yield, high puritynucleic acids. For example, a variety of publications and patentsdescribing such methods are listed herein. Examples include Short“PRODUCTION OF ENZYMES HAVING DESIRED ACTIVITIES BY MUTAGENESIS” U.S.Pat. No. 5,939,250, Thompson, et al. (1998) “METHODS FOR GENERATING ANDSCREENING NOVEL METABOLIC PATHWAYS” U.S. Pat. No. 5,824,485 and6,783,431; and Carlson, et al. (1999) “METHOD OF RECOVERING A BIOLOGICALMOLECULE FROM A RECOMBINANT MICROORGANISM” U.S. Pat. Nos. 5,908,765,5,837,470 and 5,773,221, which allege various methods for creatinglibraries from, e.g., uncharacterized heterogeneous microbial samples.The present invention provides, e.g., for automation, spatial or logicalarrays and associated tools in mediating, improving or replacing theseprocesses.

Often, effective development of a commercially relevant enzyme, proteinor biochemical pathway (e.g. for pharmaceutical or industrialapplications) involves identifying a plurality of favorable activityparameters be encoded by the candidate gene(s). Having a means ofrapidly recruiting and then diversifying a wide variety of startinggenes from a wide variety of sources—such as may share a commonstructural or activity motif—is of importance for rapid gene or pathwaydevelopment. The present application teaches the application of a familyof array operations and automated processing of a wide range ofmutagenesis, gene synthesis and recombination and technologies forimproving candidate genes.

While preliminary gene recruitment can be done by hybridization or onthe basis of logically derived and/or stored hybridization information,hybridization is often not used in confirming the activity or intactnessof a given nucleic acid within a physical array. For more refinedrecruitment or identification of promising candidate genes within anarray, it is useful to have at least one other biochemical activitymeasurement on which to contrast the various members of the storagearray. The current invention contemplates and describes a large numberof logical and laboratory-based criteria and processes for storing,maintaining and recording that information and its physical of logicallinkage with given members of the array. Thus a member of an array ismost accurately defined on the basis of its activity in each of thetests performed on it.

A wide variety of phenotypic attributes or combinations of suchattributes are useful for identifying genes for suitable for a givenapplication, process, pathway or subsequent evolution toward suchapplications. In addition to simply creating libraries from diversesamples, expressing such libraries in cells or on phage, and analyzingthe results biochemically, the present invention provides, e.g., forautomated, integrated or integrateable modules for rapidly producing andcharacterizing expression arrays, e.g., by way of in vitro transcriptionor translation tools. The present methods also describe the utility anddesign of automated processes for identifying, cataloging, selecting andsubsequently evolving genes from natural or synthetic systems.

One embodiment the present invention describes an automated process forrecruiting genes from natural, synthetic or logical sources and storinggenetic material suitable for subsequent characterization, mutagenesis,selection and evolution. In another embodiment, it describes theautomated devices or modules which carry out such processes.

In addition, the present invention describes a series of general,automatable methods for high-yield extraction of nucleic acids from awide variety of samples. In these methods, samples containing nucleicacids (e.g., as from diverse or clonal cultured or uncultured cellularpopulations; tissue sections; sera samples; samples from heterogeneousenrichment cultures, bioreactors or fermentors; samples containing oneor more uncharacterized microorganism; environmental isolates; soil,water or microcosm samples) are treated by a method, e.g., comprisingthe following processes.

First the sample is treated with a plurality of chemical lysing agents(consisting of: chaotropic substance(s), detergent(s), chelator(s),proteinase(s), exo- or endo-glucanase(s), lysozyme(s) and otherproteoglycan or cell wall degrading enzymes, etc.) under conditionswhich allow the lysing agent or agents to come into liquid contact thecell membranes the target cells. The plurality of lysing agents caninclude a chaotropic agent capable of substantially inactivating a widevariety of nucleases. Similarly, the plurality of lysing agents caninclude at least one chaotrope and at least one enzyme for lysis.Examples of lysing agents include urea, guanidine and guanidinium,enzymes, etc. Any one or more of these chemical or physical lysingconditions can be used on a given sample, or a sample may be subdividedand subjected to sequential or combinatorial lysis to: a) identifyoptimal lysing conditions, b) prepare multiple unique extracts from asingle sample and/or c) conduct parallel sample preparation, for anypurpose.

Second, the samples can be treated with at least one disruptive physicalcondition(s) or treatment(s) (e.g. freeze-thawing, freeze drying,cold-hot cycling, disruptive (rapid) mixing, sonicating, heating,incubation at pH<5.5 or >8.5, etc.). The at least one disruptivephysical condition or treatment can include incubation at a temperatureabove 37° C. and, e.g. at a temperature of >50° C. The at least onedisruptive physical condition or treatment can include at least onefreeze-thaw, mixing or sonication step and incubation at a temperatureof >50° C. The at least one disruptive physical condition or treatmentcan include at least one heating or cooling step and at least one stepwhich can cause (such as mixing, vortexing, sonicating or incubating inhypotonic media) physical shearing of cell walls and high molecularweight DNA.

The sample can be subjected to at least one physical-chemical separationstep (which may be chosen or achieve similar results such asprecipitation, solvent extraction, electrophoretic or chromatographicseparation or others) to isolate high purity nucleic acids, e.g., fromenriched cultures, natural isolates, cultured cells, tissues or sera.For example at least one alcohol mediated precipitation step or oneextraction step can be used. The use of a plurality of physico-chemicalseparation modes can be used in the extraction process. At least oneextraction step and one precipitation or chromatographic step can beused in combination.

In a preferred embodiment, the process described here is conducted underconditions in which a plurality of lysing agents and disruptive physicalagents are used on and in which the operation is integrated into anautomated device.

The automation of such a method provides a free-standing and uniquelyvaluable platform from which to conduct high throughput nucleic acidextraction and purification from diverse sample sources. Nucleic acidsprepared in such a way can be further characterized or selected, with orwithout prior cloning, by hybridization-based detection, capture (e.g.‘panning’) or direct recombination with other members of the populationor exogenous nucleic acids added to the mixture, followed by expressionscreening.

Expression screening can involve at least one in vitro transcription ortranslation step. For example, it can involve in vitro transcriptionpreceded by at least one amplification, polymerization or ligation eventin which at least one transcriptional regulatory element is operativelylinked to the nucleic acids to undergo transcription. In a presentlypreferred embodiment, the method involves the in vitro translation oflibrary members using transcripts derived from either in vitro,synthetic or cellular sources.

The present invention describes, e.g., the following automated modulesfor the isolation, detection and evolution of nucleic acids from naturaland synthetic isolates: nucleic acid isolation modules, nucleic acidgeneration modules, nucleic acid sorting or selection modules, dilutionmodules, array replication modules, expression module, screeningmodules, etc. Such modules can operate as free-standing devices or assub-elements of a larger device or other system which links one or moreof these modules physically or logically to create, modify, analyzereplicate or otherwise manipulate members of interest within the array.

The present invention also provides a logical association for organizinga multiple-phenotype screening array. For example, the present inventionprovides for detection and screening of genes in a primarily binaryprocess, where individual clones, proteins or enzymes (whether proteinor nucleic acid, or both) are identified as either having or not havinga specified property or set of properties (resulting in a binary“yes/no” logical operation by the system in evaluating the properties).In addition to strictly binary processes, degrees of activity can alsobe detected and manipulated by the system.

The invention can also include the organization of multi-phenotypescreening in which (one or more) clones in the array are described,organized, screened or otherwise sorted (in physical or computationalterms) by their activity fingerprints, such that characterization of thearray is open-ended and allows for increasingly diverse layers ofcharacterization to be applied. Such arrays can remain closed-ended withrespect to their origin or member nucleic acids. In one embodiment, thearray architecture allows for each clone, pool of clones, individual orindividual pools of nucleic acids within an array to be described inboth (or either) binary and quantitative terms with respect to a givenactivity or property and provides a means for further isolation,processing or characterization of those members selected on the basis ofeither Boolean or quantitative queries, or combinations of the two.

While not limited to these, the query-able properties include biologicalor chemical activities, physical or structural attributes, nucleic acidor amino acid sequences, source, prior processing methods, histories orexposures or physical state within the array. In another embodiment, thepresent invention provides for the automated or semi-automatedamplification, replication and in vitro transcription and/or translationof the physical array to create sub-arrays which can be stored orscreened for other properties. In preferred embodiments, the presentinvention describes a process and a device for isolating nucleic acidsfrom natural or synthetic or computational sources, storing such nucleicacids as logical (or physical) arrays based on a plurality of phenotypes(one of which may be its nucleotide sequence) and the contacting ofarrays, with one or more in vitro transcription or translation reagents.

In the present invention, the term ‘phenotype’ is used to refer to ageneral or specific set of traits for which a given clone has beenscreened. The complete complement of phenotypic traits may be deriveddirectly from laboratory data, by logical inference from such data orfrom stored databases of relevant data (e.g. such as activity, sequenceor relational databases). These traits can be directly or indirectlyscreened, including for stability under natural non-natural physical orchemical conditions, expressibility in a given cell line, strain or invitro extract, size, solubility, hybridization properties, sequence,associated regulatory elements, catalytic rate, substrate or productselectivity, luminescent, fluorescent, light scattering, x-raydiffracting, sedimentation, binding, calorimetric, refractive or otherdiverse properties.

The arrays of the invention have value in all areas in which geneproducts have utility, including pharmaceutical and chemical discoveryand manufacturing, agriculture, diagnostics, biofuels, fuel cells andbioelectronics, and many other areas. Such arrays are developed, e.g.,from gene libraries extracted from nature or natural sources. They canalso be derived computationally or via automated gene or oligonucleotidesynthesis. In addition, analogous or derivative arrays may be generatedvia the application of shuffling or other mutagenesis methods to one ormore parental nucleic acids.

While each phenotypic attribute is of value in describing a given memberof an array, certain combinations of properties can be particularlyuseful in characterizing genes for utility in pharmaceutical or chemicalmanufacturing processes. For example, an array in which at least onephysical attribute and at least one selectivity attribute are measuredfor a plurality of members of that array can be more valuable than onein which only the expression, selectivity or stability attribute hasbeen assessed.

Similarly, an array containing enzymes (or cells expressing suchenzymes) which have been quantitatively characterized for their testedfor their ability to stereoselectively convert a substrate to a givenproduct under a defined solvent or temperature regime is moreinformative to the synthetic or process chemist interested in the givenconversion than one in which only one of the properties listed has beenexamined. For synthetic and process chemistry applications physicalchemical attributes of interest include many diverse attributes. Forexample, stability or activity in solvents or mixed water-solventssystems (common solvents would, for example include polar protic andaprotic solvents, nonpolar solvents, alcohols, ethers, esters, alkanes,halogenated solvents, phenols, tetrahydrofuran, benzene and itsderivatives, aromatic, fluorinated and perfluorinated solvents, etc. . .. ), stability or activity at elevated or depressed temperatures (e.g.above 50° C. and below 20° C.; e.g., >70° C. or <10° C.), and stabilityor activity in high or low salt concentrations (e.g. >1 M or <0.050 Msodium, potassium and ammonium containing salts with chloride, bromide,nitrate, nitrite, sulfate, sulfite, carbonate, bicarbonate or amino acidcounterions). Similarly, stability or activity at high or low pressure,in oxygen-rich or oxygen deficient environments and/or in the presenceof a variety of a one or more agents capable of inactivating proteins bycovalent modification (e.g., acylating, alkylating and amide reactiveagents), stability of activity in the presence of at least phasetransfer or crosslinking agent, or stability or activity within or upona solid matrix (e.g. by covalent or noncovalent association with anatural or functionalized surface, the surface comprising a hydrophobicor hygroscopic polymer, silica, glass, metal, aluminum, alloy,cellulosic or modified cellulosic, hygroscopic insoluble material anatural biopolymer, a polysaccharide and modified forms of these) canalso be of interest.

Selectivity attributes of interest in process and combinatorialchemistry include, but are not limited to, product or substratechemoselectivity, regioselectivity, stereoselectivity andenantioselectivity; and each of these in combination with a plurality ofsolvent and physical conditions such as those described above. Thus thepresent invention describes means of making and using logical and/orphysical enzyme arrays in which each member has been characterized onthe basis of its activity under at least one nonphysiological physicalcondition and at least one selectivity attribute. For example, the atleast one nonphysiological condition can involve one or more of thefollowing conditions: a nonphysiological thermal, salt, solvent,pressure, or oxygen condition; the presence of active levels of one ormore crosslinking agents; or the presence of active levels of one ormore potential covalent modifying agents; or immobilization upon on anonbiological surface.

K. Further Embodiments

In a further aspect, the present invention provides for the use of anyapparatus, apparatus component, composition or kit herein, for thepractice of any method or assay herein, and/or for the use of anyapparatus or kit to practice any assay or method herein.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques, methods, compositions,apparatus and systems described above may be used in variouscombinations. All publications, patents, patent documents (includingpatent applications) and other references cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent document orother reference were individually indicated to be incorporated byreference for all purposes.

1-99. (canceled)
 100. A diversity generation device, comprising: (i) aprogrammed thermocycler that receives nucleic acid fragments from (ii)and reassembles them to produce shuffled nucleic acids, wherein theprogrammed thermocycler comprises a thermocycler operably coupled to acomputer, wherein the computer comprises one or more instruction setthat does one or more of the following: calculates an amount of uraciland an amount of thymidine for use in the programmed thermocycler orcalculates one or more crossover region between two or more parentalnucleic acid sequence; and (ii) a fragmentation module operably coupledto the programmed thermocycler, wherein the fragmentation modulefragments parental nucleic acids.
 101. (canceled)
 102. The diversitygeneration device of claim 100, wherein the one or more instruction setreceives user input data and sets up one or more cycle to be performedby the programmed thermocycler.
 103. The diversity generation device ofclaim 102, wherein the input data comprises one or more of: one or moreparental nucleic acid nucleic acid sequence, a desired crossoverfrequency, an extension temperature, or an annealing temperature. 104.The diversity generation device of claim 100, wherein the one or moreinstruction set calculates the amount of uracil and the amount ofthymidine based on a desired fragment size.
 105. The diversitygeneration device of claim 102, wherein the one or more instruction setdirects the one or more cycle on the diversity generation device, whichone or more cycle: (a) amplifies the one or more parental nucleic acidsequence; (b) fragments the one or more parental nucleic acid sequenceto produce one or more nucleic acid fragment; (c) reassembles the one ormore nucleic acid fragment to produce one or more shuffled nucleic acid;and, (d) amplifies the one or more shuffled nucleic acid.
 106. Thediversity generation device of claim 105, wherein step (a) comprisesamplifying the one or more parental nucleic acid sequence in thepresence of uracil.
 107. The diversity generation device of claim 105,wherein the one or more cycle pauses between step (a) and step (b) toallow addition of one or more fragmentation reagent.
 108. The diversitygeneration device of claim 100, wherein the one or more instruction setperforms one or more calculation based on one or more theoreticalprediction of a nucleic acid melting temperature or on one or more setof empirical data, which empirical data comprises a comparison of one ormore nucleic acid melting temperature.
 109. The diversity generationdevice of claim 105, wherein the one or more instruction set instructsthe fragmentation module to fragment the parental nucleic acids toproduce one or more nucleic acid fragments having a desired meanfragment size.
 110. The diversity generation device of claim 100,wherein the computer receives user input data that comprises parentalnucleic acid sequences, and wherein the programmed thermocyclercomprises software for performing one or more shuffling calculations onthe parental nucleic acid sequences, which software is embodied on a webpage or is installed directly in the thermocycler.
 111. The diversitygeneration device of claim 100, wherein the fragmentation modulefragments one or more parental nucleic acids by sonication, DNase IIdigestion, random primer extension, or uracil incorporation andtreatment with one or more uracil cleavage enzyme.
 112. A diversitygeneration device comprising: (i) a computer, which computer comprisesat least a first instruction set for creating nucleic acid fragmentsequences from one or more parental nucleic acid sequence, wherein thefirst instruction set limits or expands diversity encoded by one or morenucleic acid fragment; (ii) a synthesizer module operably coupled to thecomputer, which synthesizer module synthesizes the one or more nucleicacid fragment sequence; and, (iii) a thermocycler operably coupled tothe synthesizer, which thermocycler generates diverse sequences from thenucleic acid fragment sequences.
 113. The diversity generation device ofclaim 112, wherein the first instruction set limits or expands diversityencoded by one or more nucleic acid fragment sequence by adding orremoving one or more nucleotides encoding similar diversity; selectingnucleotides encoding a frequently used amino acid at one or morespecific position; using one or more sequence activity calculation;using a calculated overlap with one or more additional oligonucleotide;based on an amount of degeneracy, or based on a melting temperature.114. The diversity generation device of claim 112, wherein thesynthesizer module comprises a microarray oligonucleotide synthesizer.115. The diversity generation device of claim 114, wherein thesynthesizer module comprises an ink-jet printer head basedoligonucleotide synthesizer.
 116. The diversity generation device ofclaim 112, wherein the synthesizer module synthesizes the one or morenucleic acid fragment sequences on a solid support.
 117. The diversitygeneration device of claim 112, wherein the synthesizer module uses oneor more mononucleotide coupling reactions or one or more trinucleotidecoupling reactions to synthesize the one or more nucleic acid fragmentsequence.
 118. The diversity generation device of claim 112, wherein thethermocycler performs an assembly/rescue PCR reaction.
 119. Thediversity generation device of claim 118, wherein the computer comprisesat least a second instruction set, which second instruction setdetermines at least a first set of conditions for the assembly/rescuePCR reaction.
 120. The diversity generation device of claim 112, thedevice further comprising a screening module for screening the diversesequences for a desired characteristic.
 121. The diversity generationdevice of claim 120, wherein the screening module comprises ahigh-throughput screening module. 122-299. (canceled)